Compounds including an anti-inflammatory pharmacore and methods of use

ABSTRACT

This invention provides novel compounds comprising the following anti-inflammatory pharmacore: 
     
       
         
         
             
             
         
       
     
     wherein X, R 1  and R 2  are defined herein. Also provided are pharmaceutical compositions, kits and articles of manufacture comprising such compounds, methods and intermediates useful for making the compounds, and methods of using the compounds and compositions.

The present application claims the benefit of priority to U.S.Provisional Application No. 61/046,363, filed Apr. 18, 2008, the entirecontents of this application being incorporated by reference.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of biology andmedicine. More particularly, it concerns compounds and methods for thetreatment and prevention of diseases such as those associated withoxidative stress and inflammation.

II. Description of Related Art

Many serious and intractable human diseases are associated withdysregulation of inflammatory processes, including diseases such ascancer, atherosclerosis, and diabetes, which were not traditionallyviewed as inflammatory conditions. Similarly, autoimmune diseases suchas rheumatoid arthritis, lupus, psoriasis, and multiple sclerosisinvolve inappropriate and chronic activation of inflammatory processesin affected tissues, arising from dysfunction of self vs. non-selfrecognition and response mechanisms in the immune system. Inneurodegenerative diseases such as Alzheimer's and Parkinson's diseases,neural damage is correlated with activation of microglia and elevatedlevels of pro-inflammatory proteins such as inducible nitric oxidesynthase (iNOS).

One aspect of inflammation is the production of inflammatoryprostaglandins such as prostaglandin E, whose precursors are produced byCOX-2. High levels of COX-2 are found in inflamed tissues. Consequently,inhibition of COX-2 is known to reduce many symptoms of inflammation anda number of important anti-inflammatory drugs (e.g., ibuprofen andcelecoxib) act by inhibiting COX-2 activity. Recent research, however,has demonstrated that a class of cyclopentenone prostaglandins (e.g.,15-deoxy prostaglandin J2, a.k.a. PGJ2) plays a role in stimulating theorchestrated resolution of inflammation. COX-2 is also associated withthe production of cyclopentenone prostaglandins. Consequently,inhibition of COX-2 may interfere with the full resolution ofinflammation, potentially promoting the persistence of activated immunecells in tissues and leading to chronic, “smoldering” inflammation. Thiseffect may be responsible for the increased incidence of cardiovasculardisease in patients using selective COX-2 inhibitors for long periods oftime. Corticosteroids, another important class of anti-inflammatorydrugs, have many undesirable side effects and frequently are notsuitable for chronic use. Newer protein-based drugs, such as anti-TNFmonoclonal antibodies, have proven to be effective for the treatment ofcertain autoimmune diseases such as rheumatoid arthritis. However, thesecompounds must be administered by injection, are not effective in allpatients, and may have severe side effects. In many severe forms ofinflammation (e.g., sepsis, acute pancreatitis), existing drugs areineffective. In addition, currently available drugs do not havesignificant antioxidant properties, and are not effective in reducingoxidative stress associated with excessive production of reactive oxygenspecies and related molecules such as peroxynitrite. Accordingly, thereis a pressing need for improved therapeutics with antioxidant andanti-inflammatory properties.

Synthetic triterpenoid analogs of oleanolic acid have been shown to beinhibitors of cellular inflammatory processes, such as the induction byIFN-γ of inducible nitric oxide synthase (iNOS) and of COX-2 in mousemacrophages. See Honda et al. (2000a); Honda et al. (2000b), and Hondaet al. (2002), which are all incorporated herein by reference. Forexample, one of these, 2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acidmethyl ester (CDDO-Me), is currently in clinical trials for a variety ofdisorders related to inflammation, including cancer and diabeticnephropathy. Synthetic derivatives of another triterpenoid, betulinicacid, have also been shown to inhibit cellular inflammatory processes,although these compounds have been less extensively characterized (Hondaet al., Bioorg Med Chem Lett. 2006; 16(24):6306-9). The pharmacology ofthese synthetic triterpenoid molecules is complex. Compounds derivedfrom oleanolic acid have been shown to affect the function of multipleprotein targets and thereby modulate the activity of several importantcellular signaling pathways related to oxidative stress, cell cyclecontrol, and inflammation (e.g., Dinkova-Kostova et al., Proc Natl AcadSci USA. 2005; 102(12):4584-9; Ahmad et al., J. Biol Chem. 2006;281(47):35764-9; Ahmad et al., Cancer Res. 2008; 68(8):2920-6; Liby etal., Nat Rev Cancer. 2007; 7(5):357-69). Derivatives of betulinic acid,though they have shown comparable anti-inflammatory properties, alsoappear to have significant differences in their pharmacology compared toOA-derived compounds (Liby et al., Mol Cancer Ther 2007; 6(7)). Further,it is not certain that the triterpenoid starting materials employed todate have optimal properties compared to other possible startingmaterials. Given that the biological activity profiles of knowntriterpenoid derivatives vary, and in view of the wide variety ofdiseases that may be treated or prevented with compounds having potentantioxidant and anti-inflammatory effects, and the high degree of unmetmedical need represented within this variety of diseases, it isdesirable to synthesize new compounds with diverse structures that mayhave improved biological activity profiles for the treatment of one ormore indications.

SUMMARY OF THE INVENTION

The present disclosure overcomes limitation of the prior art byproviding new compounds with antioxidant and anti-inflammatoryproperties, methods for their manufacture, and methods for their use.

In some aspects, the disclosure provides compounds comprising:

-   a) the basic skeleton of an organic compound having two to eight    five and/or six-membered rings provided that the basic skeleton is    not the basic skeleton of argentatin, betulinic acid, lanostane,    oleanic acid, boswellic acid, glycyrrhetinic acid, ursolic acid, or    tricyclic-bis-enone;-   b) a structural unit of the formula:

-    wherein:    -   the carbon atoms labeled 1, 2 and 3 are part of a five or        six-membered ring;    -   X is cyano or —C(O)R_(a), wherein R_(a) is:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, azido or            mercapto; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), alkoxy_((C≦12)),            alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylsilyloxy_((C≦12)), or substituted            versions of any of these groups; and    -   R₁ and R₂ are each independently:        -   hydrogen; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            arylamino_((C≦12)), aralkylamino_((C≦12)),            heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),            amido_((C≦12)), or a substituted version of any of these            groups; or    -   R₁ and R₂ are taken together and are alkanediyl_((C≦12)),        alkenediyl_((C≦12)), alkanediyl_((C≦12)) or alkenediyl_((C≦12));        and-   c) from 0 to 8 chemical groups attached to a carbon atom of the    basic skeleton other than carbon atoms 1, 2, 3 or 4, wherein each    chemical group is independently:    -   hydroxy, halo, oxo, amino, hydroxyamino, nitro, imino, cyano,        azido, mercapto, or thio; or    -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),        aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),        heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),        alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),        aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),        heteroaralkoxy_((C≦12)), acyloxy_((C≦12)), alkylamino_((C≦12)),        dialkylamino_((C≦12)), alkoxyamino_((C≦12)),        alkenylamino_((C≦12)), alkynylamino_((C≦12)),        arylamino_((C≦12)), aralkylamino_((C≦12)),        heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),        alkylsulfonylamino_((C≦12)), amido_((C≦12)),        alkylimino_((C≦12)), alkenylimino_((C≦12)),        alkynylimino_((C≦12)), arylimino_((C≦12)),        aralkylimino_((C≦12)), heteroarylimino_((C≦12)),        heteroaralkylimino_((C≦12)), acylimino_((C≦12)),        alkylthio_((C≦12)), alkenylthio_((C≦12)), alkynylthio_((C≦12)),        arylthio_((C≦12)), aralkylthio_((C≦12)),        heteroarylthio_((C≦12)), heteroaralkylthio_((C≦12)),        acylthio_((C≦12)), thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),        alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),        arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),        heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),        alkylammonium_((C≦12)), alkylsulfonium_((C≦12)),        alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a substituted        version of any of these groups;        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, acetals, ketals, prodrugs, or optical        isomers thereof.

In some embodiments, R₁ and R₂ are each independently hydrogen,alkyl_((C≦8)) or substituted alkyl_((C≦8)). In some embodiments, R₁ andR₂ are both hydrogen. In some embodiments, R₁ and R₂ are both methyl. Insome embodiments, R₁ and R₂ are both not hydrogen. In some embodiments,the organic compound is not a triterpenoid.

In some aspects, the disclosure provides compounds comprising:

-   a) the basic skeleton of a natural product having two to eight    rings, provided that the natural product is not argentatin,    betulinic acid, lanostane, oleanic acid, or ursolic acid;-   b) a structural unit of the formula:

-    wherein:    -   the carbon atoms labeled 1, 2 and 3 are part of a six-membered        ring;    -   X is cyano, fluoroalkyl_((C≦8)), substituted,        fluoroalkyl_((C≦8)), or —C(O)R_(a) wherein R_(a) is:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, azido or            mercapto; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), alkoxy_((C≦12)),            alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylsilyloxy_((C≦12)), or substituted            versions of any of these groups; and    -   R₁ and R₂ are each independently:        -   hydrogen; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            arylamino_((C≦12)), aralkylamino_((C≦12)),            heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),            amido_((C≦12)), or a substituted version of any of these            groups; or    -   R₁ and R₂ are taken together and are alkanediyl_((C≦12)),        alkenediyl_((C≦12)), alkanediyl_((C≦12)) or alkenediyl_((C≦12));        and-   c) from 0 to 8 chemical groups attached to a carbon atom of the    basic skeleton other than carbon atoms 1, 2, 3 or 4, wherein each    chemical group is independently:    -   hydroxy, halo, oxo, amino, hydroxyamino, nitro, imino, cyano,        azido, mercapto, or thio; or    -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),        aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),        heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),        alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),        aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),        heteroaralkoxy_((C≦12)), acyloxy_((C≦12)), alkoxyamino_((C≦12)),        alkylamino_((C≦12)), dialkylamino_((C≦12)),        alkenylamino_((C≦12)), alkynylamino_((C≦12)),        arylamino_((C≦12)), aralkylamino_((C≦12)),        heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),        alkylsulfonylamino_((C≦12)), amido_((C≦12)),        alkylimino_((C≦12)), alkenylimino_((C≦12)),        alkynylimino_((C≦12)), arylimino_((C≦12)),        aralkylimino_((C≦12)), heteroarylimino_((C≦12)),        heteroaralkylimino_((C≦12)), acylimino_((C≦12)),        alkylthio_((C≦12)), alkenylthio_((C≦12)), alkynylthio_((C≦12)),        arylthio_((C≦12)), aralkylthio_((C≦12)),        heteroarylthio_((C≦12)), heteroaralkylthio_((C≦12)),        acylthio_((C≦12)), thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),        alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),        arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),        heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),        alkyl-ammonium_((C≦12)), alkylsulfonium_((C≦12)),        alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a substituted        version of any of these groups;        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, acetals, ketals, prodrugs, or optical        isomers thereof.

In some embodiments, R₁ and R₂ are each independently hydrogen,alkyl_((C≦8)) or substituted alkyl_((C≦8)). In some embodiments, R₁ andR₂ are both hydrogen. In some embodiments, R₁ and R₂ are both methyl. Insome embodiments, R₁ and R₂ are both not hydrogen. In some embodiments,the natural product is not boswellic acid or glycyrrhetinic acid. Insome embodiments, the natural product is not a triterpenoid. In someembodiments, X is —CN or —C(═O)NHS(═O)₂CH₃. In some embodiments, X is—CN. In some embodiments, X is fluoroalkyl_((C≦8)). In some embodiments,X is —CF₃.

In some embodiments, the structural unit is further defined as:

In some embodiments, the basic skeleton of the natural product has tworings. In some embodiments, the rings are connected to one another by asingle chain of atoms. In some embodiments, the backbone of the singlechain further comprises at least one carbon-carbon double bond.

In some embodiments, the natural product is curcumin. In someembodiments, the compound is further defined by the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydrogen; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            arylamino_((C≦12)), aralkylamino_((C≦12)),            heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),            amido_((C≦12)), or a substituted version of any of these            groups; or    -   R₁ and R₂ are taken together and are alkanediyl_((C≦12)),        alkenediyl_((C≦12)), alkanediyl_((C≦12)); or        alkenediyl_((C≦12)); and    -   R₃ and R₄ are each independently:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,            azido, or mercapto; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), amido_((C≦12)),            alkylsulfonylamino_((C≦12)), alkylsilyl_((C≦12)),            alkylsilyloxy_((C≦12)), or a substituted version of any of            these groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, acetals, ketals, prodrugs, or optical            isomers thereof. In some variations, R₁ and R₂ are both            methyl.            For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof. For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the natural product is resveratrol. In someembodiments, the compound is further defined by the formula:

wherein:

-   R₁ and R₂ are each independently:    -   hydrogen; or    -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),        aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),        heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),        aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),        heteroaralkoxy_((C≦12)), acyloxy_((C≦12)), alkylamino_((C≦12)),        dialkylamino_((C≦12)), arylamino_((C≦12)),        aralkylamino_((C≦12)), heteroarylamino_((C≦12)),        heteroaralkylamino_((C≦12)), amido_((C≦12)), or a substituted        version of any of these groups; or-   R₁ and R₂ are taken together and are alkanediyl_((C≦12)),    alkenediyl_((C≦12)), alkanediyl_((C≦12)) or alkenediyl_((C≦12)); and-   R₅ and R₆ are each independently:    -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,        azido, or mercapto; or    -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),        aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),        heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),        alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),        aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),        heteroaralkoxy_((C≦12)), acyloxy_((C≦12)), alkylamino_((C≦12)),        dialkylamino_((C≦12)), alkoxyamino_((C≦12)),        alkenylamino_((C≦12)), alkynylamino_((C≦12)),        arylamino_((C≦12)), aralkylamino_((C≦12)),        heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),        amido_((C≦12)), alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or        a substituted version of any of these groups;        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, acetals, ketals, prodrugs, or optical        isomers thereof. In some embodiments, R₁ and R₂ are both methyl.        For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the basic skeleton of the natural product has threerings. In some embodiments, the natural product is gallocatechol. Insome embodiments, the compound is further defined by the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydrogen; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            arylamino_((C≦12)), aralkylamino_((C≦12)),            heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),            amido_((C≦12)), or a substituted version of any of these            groups; or    -   R₁ and R₂ are taken together and are alkanediyl_((C≦12)),        alkenediyl_((C≦12)), alkanediyl_((C≦12)) or alkenediyl_((C≦12));    -   R₇, R₈ and R₉ are each independently:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,            azido, or mercapto; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)),            heteroaryl-heteroaralkylamino_((C≦12)), amido_((C≦12)),            alkylsilyl_((C≦12)), amino_((C≦12)), alkylsilyloxy_((C≦12)),            or a substituted version of any of these groups; and    -   R₁₀ is:        -   hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro,            imino, cyano, azido, mercapto, or thio; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),            alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylimino_((C≦12)), alkenylimino_((C≦12)),            alkynylimino_((C≦12)), arylimino_((C≦12)),            aralkylimino_((C≦12)), heteroarylimino_((C≦12)),            heteroaralkylimino_((C≦12)), acylimino_((C≦12)),            alkylthio_((C≦12)), alkenylthio_((C≦12)),            alkynylthio_((C≦12)), arylthio_((C≦12)),            aralkylthio_((C≦12)), heteroarylthio_((C≦12)),            heteroaralkylthio_((C≦12)), acylthio_((C≦12)),            thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),            alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),            arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),            heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),            alkyl-ammonium_((C≦12)), alkylsulfonium_((C≦12)),            alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a            substituted version of any of these groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, acetals, ketals, prodrugs, or optical            isomers thereof. In some embodiments, R₁ and R₂ are both            methyl. For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the compound is further defined by the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydrogen; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            arylamino_((C≦12)), aralkylamino_((C≦12)),            heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),            amido_((C≦12)), or a substituted version of any of these            groups; or    -   R₁ and R₂ are taken together and are alkanediyl_((C≦12)),        alkenediyl_((C≦12)), alkanediyl_((C≦12)) or alkenediyl_((C≦12));    -   R₁₁ is:        -   hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro,            imino, cyano, azido, mercapto, or thio; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),            alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylimino_((C≦12)), alkenylimino_((C≦12)),            alkynylimino_((C≦12)), arylimino_((C≦12)),            aralkylimino_((C≦12)), heteroarylimino_((C≦12)),            heteroaralkylimino_((C≦12)), acylimino_((C≦12)),            alkylthio_((C≦12)), alkenylthio_((C≦12)),            alkynylthio_((C≦12)), arylthio_((C≦12)),            aralkylthio_((C≦12)), heteroarylthio_((C≦12)),            heteroaralkylthio_((C≦12)), acylthio_((C≦12)),            thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),            alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),            arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),            heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),            alkyl-ammonium_((C≦12)), alkylsulfonium_((C≦12)),            alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a            substituted version of any of these groups; and    -   R₁₂, R₁₃ and R₁₄ are each independently:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,            azido, or mercapto; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), amido_((C≦12)),            alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a            substituted version of any of these groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, acetals, ketals, prodrugs, or optical            isomers thereof. In some embodiments, R₁ and R₂ are both            methyl. For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the basic skeleton of the natural product has fourrings. In other embodiments, the basic skeleton of the natural producthas five rings. In some embodiments, the natural product is celastrol.In further embodiments, the basic skeleton of the natural product hassix rings. In some embodiments, the natural product is hecogenin,tigogenin, or sarsapogenin.

In some embodiments, the compound is further defined by the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydrogen; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            arylamino_((C≦12)), aralkylamino_((C≦12)),            heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),            amido_((C≦12)), or a substituted version of any of these            groups; or    -   R₁ and R₂ are taken together and are alkanediyl_((C≦12)),        alkenediyl_((C≦12)), alkanediyl_((C≦12)) or alkenediyl_((C≦12));        and    -   R₁₅, R₁₆, R₁₇, R₁₈ and R₁₉ are each independently:        -   hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro,            imino, cyano, azido, mercapto, or thio; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),            alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkoxyamino_((C≦12)), alkylamino_((C≦12)),            dialkylamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylimino_((C≦12)), alkenylimino_((C≦12)),            alkynylimino_((C≦12)), arylimino_((C≦12)),            aralkylimino_((C≦12)), heteroarylimino_((C≦12)),            heteroaralkylimino_((C≦12)), acylimino_((C≦12)),            alkylthio_((C≦12)), alkenylthio_((C≦12)),            alkynylthio_((C≦12)), arylthio_((C≦12)),            aralkylthio_((C≦12)), heteroarylthio_((C≦12)),            heteroaralkylthio_((C≦12)), acylthio_((C≦12)),            thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),            alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),            arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),            heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),            alkyl-ammonium_((C≦12)), alkylsulfonium_((C≦12)),            alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a            substituted version of any of these groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, acetals, ketals, prodrugs, or optical            isomers thereof. In some embodiments, R₁ and R₂ are both            methyl. In some embodiments, R₁₇ is methyl. For example, the            disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In another aspect, the disclosure provides compounds of the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydrogen; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            arylamino_((C≦12)), aralkylamino_((C≦12)),            heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),            amido_((C≦12)), or a substituted version of any of these            groups; or    -   R₁ and R₂ are taken together and are alkanediyl_((C≦12)),        alkenediyl_((C≦12)), alkanediyl_((C≦12)) or alkenediyl_((C≦12));        and    -   R₂₀, R₂₁, and R₂₂ are each independently:        -   hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro,            imino, cyano, azido, mercapto, or thio; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),            alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkoxyamino_((C≦12)), alkylamino_((C≦12)),            dialkylamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylimino_((C≦12)), alkenylimino_((C≦12)),            alkynylimino_((C≦12)), arylimino_((C≦12)),            aralkylimino_((C≦12)), heteroarylimino_((C≦12)),            heteroaralkylimino_((C≦12)), acylimino_((C≦12)),            alkylthio_((C≦12)), alkenylthio_((C≦12)),            alkynylthio_((C≦12)), arylthio_((C≦12)),            aralkylthio_((C≦12)), heteroarylthio_((C≦12)),            heteroaralkylthio_((C≦12)), acylthio_((C≦12)),            thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),            alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),            arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),            heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),            alkyl-ammonium_((C≦12)), alkylsulfonium_((C≦12)),            alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a            substituted version of any of these groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, acetals, ketals, prodrugs, or optical            isomers thereof. In some embodiments, R₁ and R₂ are both            hydrogen. In other embodiments, R₁ and R₂ are not hydrogen.            In still other embodiments, R₁ and R₂ are both methyl.

In some embodiments, the compound is further defined as:

wherein R₂₀, R₂₁, and R₂₂ are each independently:

-   -   hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro, imino,        cyano, azido, mercapto, or thio; or    -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),        aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),        heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),        alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),        aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),        heteroaralkoxy_((C≦12)), acyloxy_((C≦12)), alkylamino_((C≦12)),        dialkylamino_((C≦12)), alkoxyamino_((C≦12)),        alkenylamino_((C≦12)), alkynylamino_((C≦12)),        arylamino_((C≦12)), aralkylamino_((C≦12)),        heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),        alkylsulfonylamino_((C≦12)), amido_((C≦12)),        alkylimino_((C≦12)), alkenylimino_((C≦12)),        alkynylimino_((C≦12)), arylimino_((C≦12)),        aralkylimino_((C≦12)), heteroarylimino_((C≦12)),        heteroaralkylimino_((C≦12)), acylimino_((C≦12)),        alkylthio_((C≦12)), alkenylthio_((C≦12)), alkynylthio_((C≦12)),        arylthio_((C≦12)), aralkylthio_((C≦12)),        heteroarylthio_((C≦12)), heteroaralkylthio_((C≦12)),        acylthio_((C≦12)), thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),        alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),        arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),        heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),        alkyl-ammonium_((C≦12)), alkylsulfonium_((C≦12)),        alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a substituted        version of any of these groups;        or pharmaceutically acceptable salts, esters, hydrates,        solvates, tautomers, acetals, ketals, prodrugs, or optical        isomers thereof.

For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the disclosure provides compounds selected from thegroup consisting of:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some variations, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some variations, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In another aspect, the disclosure provides compounds of the formula:

wherein:

-   -   R₂₃ and R₂₄ are each independently:        -   or hydrogen, alkyl_((C≦8)) or substituted alkyl_((C≦8)); or    -   R₂₃ and R₂₄ are taken together and are alkanediyl_((C≦12)),        alkenediyl_((C≦12)), alkanediyl_((C≦12)) or alkenediyl_((C≦12));    -   R₂₅, R₂₆ and R₂₇ are each independently:        -   hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro,            imino, cyano, azido, mercapto, or thio; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),            alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),            aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkoxyamino_((C≦12)), alkylamino_((C≦12)),            dialkylamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylimino_((C≦12)), alkenylimino_((C≦2)),            alkynylimino_((C≦12)), arylimino_((C≦12)),            aralkylimino_((C≦12)), heteroarylimino_((C≦12)),            heteroaralkylimino_((C≦12)), acylimino_((C≦12)),            alkylthio_((C≦12)), alkenylthio_((C≦12)),            alkynylthio_((C≦12)), arylthio_((C≦12)),            aralkylthio_((C≦12)), heteroarylthio_((C≦12)),            heteroaralkylthio_((C≦12)), acylthio_((C≦12)),            thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),            alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),            arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),            heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),            alkylammonium_((C≦12)), alkylsulfonium_((C≦12)),            alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a            substituted version of any of these groups;            or pharmaceutically acceptable salts, esters, hydrates,            solvates, tautomers, acetals, ketals, prodrugs, or optical            isomers thereof.

For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

For example, the disclosure provides:

or pharmaceutically acceptable salts, hydrates, solvates, tautomers, oroptical isomers thereof.

In some embodiments, the present disclosure provides compounds selectedfrom the group consisting of:

-   (S)-3-((1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-4,4-dimethyl-3,5-dioxohepta-1,6-dienyl)-3,5,5-trimethyl-6-oxocyclohex-1-enecarbonitrile,-   (S)-3-((1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-5,5-dimethoxy-4,4-dimethyl-3-oxohepta-1,6-dienyl)-3,5,5-trimethyl-6-oxocyclohex-1-enecarbonitrile,-   (S)-3-((E)-4-(2-(4-hydroxy-3-methoxystyryl)-1,3-dioxolan-2-yl)-4-methyl-3-oxopent-1-enyl)-3,5,5-trimethyl-6-oxocyclohex-1-enecarbonitrile,-   (S)-3-((E)-4-(2-(4-(tert-butyldimethylsilyloxy)-3-methoxystyryl)-1,3-dioxolan-2-yl)-4-methyl-3-oxopent-1-enyl)-3,5,5-trimethyl-6-oxocyclohex-1-enecarbonitrile,-   (S,E)-3-(4-hydroxystyryl)-3,5,5-trimethyl-6-oxocyclohex-1-enecarbonitrile,-   (2S,3S)-6-cyano-4-a,8,8-trimethyl-7-oxo-2-(3,4,5-trihydroxyphenyl)-3,4,4a,7,8,8a-hexahydro-2H-chromen-3-yl3,4,5-trihydroxybenzoate,    and-   (2R,3R)-7-cyano-5,5,8a-trimethyl-6-oxo-2-(3,4,5-trihydroxyphenyl)-3,4,4a,5,6,8a-hexahydro-2H-chromen-3-yl3,4,5-trihydroxybenzoate.

In some embodiments, the present disclosure provides compounds selectedfrom the group consisting of:

-   (S,E)-3-(4-hydroxystyryl)-3-methyl-6-oxocyclohex-1-enecarbonitrile,    and-   (3S)-3-(4-hydroxystyryl)-3,5-dimethyl-6-oxocyclohex-1-enecarbonitrile.

In some embodiments, the present disclosure provides compounds selectedfrom the group consisting of:

-   (10S,13S)-4,4,10,13-tetramethyl-3,17-dioxo-4,5,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthrene-2-carbonitrile,-   (10S,13S,17S)-17-hydroxy-4,4,10,13-tetramethyl-3-oxo-4,5,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthrene-2-carbonitrile,    and-   (10S,13S)-4,4,10,13-tetramethyl-3-oxo-17-(trimethylsilyloxy)-4,5,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthrene-2-carbonitrile.

In some embodiments, the present disclosure provides compounds selectedfrom the group consisting of:

-   (10S,13R)-17-(methoxymethoxy)-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-4-carbonitrile,    and-   (10S,13R)-17-hydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-4-carbonitrile.

In some embodiments, compounds of the present disclosure are in the formof pharmaceutically acceptable salts. In other embodiments, compounds ofthe present disclosure are not be in the form of a pharmaceuticallyacceptable salts.

In some embodiments, compounds of the present disclosure are esters ofthe above formulas. The ester may, for example, result from acondensation reaction between a hydroxy group of the formula and thecarboxylic acid group of biotin.

In some embodiments, the compounds of the present disclosure are presentas mixtures of stereoisomers. In other embodiments, the compounds of thepresent disclosure are present as single stereoisomers.

In some embodiments, compounds of the present disclosure may be used asinhibitors of IFN-γ-induced nitrous oxide (NO) production inmacrophages, for example, having an IC₅₀ value of less than 0.2 μM.

Other general aspects of the present disclosure contemplate apharmaceutical composition comprising as an active ingredient a compoundof the present disclosure and a pharmaceutically acceptable carrier. Thecomposition may, for example, be adapted for administration by a routeselected from the group consisting of orally, intraadiposally,intraarterially, intraarticularly, intracranially, intradermally,intralesionally, intra-muscularly, intranasally, intraocularally,intrapericardially, intraperitoneally, intra-pleurally,intraprostaticaly, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, lipo-somally, locally, mucosally,orally, parenterally, rectally, subconjunctival, subcutaneously,sublingually, topically, transbuccally, transdermally, vaginally, incrèmes, in lipid compositions, via a catheter, via a lavage, viacontinuous infusion, via infusion, via inhalation, via injection, vialocal delivery, via localized perfusion, bathing target cells directly,or any combination thereof. In particular embodiments, the compositionmay be formulated for oral delivery. In particular embodiments, thecomposition is formulated as a hard or soft capsule, a tablet, a syrup,a suspension, a wafer, or an elixir. In certain embodiments, the softcapsule is a gelatin capsule. Certain compositions may comprise aprotective coating, such as those compositions formulated for oraldelivery. Certain compositions further comprise an agent that delaysabsorption, such as those compositions formulated for oral delivery.Certain compositions may further comprise an agent that enhancessolubility or dispersibility, such as those compositions formulated fororal delivery. Certain compositions may comprise a compound of thepresent disclosure, wherein the compound is dispersed in a liposome, anoil and water emulsion or a water and oil emulsion.

Yet another general aspect of the present disclosure contemplates atherapeutic method comprising administering a pharmaceutically effectivecompound of the present disclosure to a subject. The subject may, forexample, be a human. These or any other methods of the presentdisclosure may further comprise identifying a subject in need oftreatment.

Another method of the present disclosure contemplates a method oftreating cancer in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure. The cancer may be any type of cancer, such as a carcinoma,sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma,or seminoma. Other types of cancers include cancer of the bladder,blood, bone, brain, breast, central nervous system, colon, endometrium,esophagus, genitourinary tract, head, larynx, liver, lung, neck, ovary,pancreas, prostate, spleen, small intestine, large intestine, stomach,or testicle. In these or any other methods, the subject may be aprimate. This or any other method may further comprise identifying asubject in need of treatment. The subject may have a family or patienthistory of cancer. In certain embodiments, the subject has symptoms ofcancer. The compounds of the invention may be administered via anymethod described herein, such as locally. In certain embodiments, thecompound is administered by direct intratumoral injection or byinjection into tumor vasculature. In certain embodiments, the compoundsmay be administered systemically. The compounds may be administeredintravenously, intraarterially, intramuscularly, intraperitoneally,subcutaneously or orally, in certain embodiments.

In certain embodiments regarding methods of treating cancer in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure, thepharmaceutically effective amount is 0.1-1000 mg/kg. In certainembodiments, the pharmaceutically effective amount is administered in asingle dose per day. In certain embodiments, the pharmaceuticallyeffective amount is administered in two or more doses per day. Thecompound may be administered by contacting a tumor cell during ex vivopurging, for example. The method of treatment may comprise any one ormore of the following: a) inducing cytotoxicity in a tumor cell; b)killing a tumor cell; c) inducing apoptosis in a tumor cell; d) inducingdifferentiation in a tumor cell; or e) inhibiting growth in a tumorcell. The tumor cell may be any type of tumor cell, such as a leukemiacell. Other types of cells include, for example, a bladder cancer cell,a breast cancer cell, a lung cancer cell, a colon cancer cell, aprostate cancer cell, a liver cancer cell, a pancreatic cancer cell, astomach cancer cell, a testicular cancer cell, a brain cancer cell, anovarian cancer cell, a lymphatic cancer cell, a skin cancer cell, abrain cancer cell, a bone cancer cell, or a soft tissue cancer cell.

Combination treatment therapy is also contemplated by the presentdisclosure. For example, regarding methods of treating cancer in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure, the method mayfurther comprise a treatment selected from the group consisting ofadministering a pharmaceutically effective amount of a second drug,radiotherapy, gene therapy, and surgery. Such methods may furthercomprise (1) contacting a tumor cell with the compound prior tocontacting the tumor cell with the second drug, (2) contacting a tumorcell with the second drug prior to contacting the tumor cell with thecompound, or (3) contacting a tumor cell with the compound and thesecond drug at the same time. The second drug may, in certainembodiments, be an antibiotic, anti-inflammatory, anti-neoplastic,anti-proliferative, anti-viral, immunomodulatory, or immunosuppressive.The second drug may be an alkylating agent, androgen receptor modulator,cytoskeletal disruptor, estrogen receptor modulator, histone-deacetylaseinhibitor, HMG-CoA reductase inhibitor, prenyl-protein transferaseinhibitor, retinoid receptor modulator, topoisomerase inhibitor, ortyrosine kinase inhibitor. In certain embodiments, the second drug is5-azacitidine, 5-fluorouracil, 9-cis-retinoic acid, actinomycin D,alitretinoin, all-trans-retinoic acid, annamycin, axitinib, belinostat,bevacizumab, bexarotene, bosutinib, busulfan, capecitabine, carboplatin,carmustine, CD437, cediranib, cetuximab, chlorambucil, cisplatin,cyclophosphamide, cytarabine, dacarbazine, dasatinib, daunorubicin,decitabine, docetaxel, dolastatin-10, doxifluridine, doxorubicin,doxorubicin, epirubicin, erlotinib, etoposide, etoposide, gefitinib,gemcitabine, gemtuzumab ozogamicin, hexamethylmelamine, idarubicin,ifosfamide, imatinib, irinotecan, isotretinoin, ixabepilone, lapatinib,LBH589, lomustine, mechlorethamine, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, MS-275, neratinib, nilotinib,nitrosourea, oxaliplatin, paclitaxel, plicamycin, procarbazine,semaxanib, semustine, sodium butyrate, sodium phenylacetate,streptozotocin, suberoylanilide hydroxamic acid, sunitinib, tamoxifen,teniposide, thiopeta, tioguanine, topotecan, TRAIL, trastuzumab,tretinoin, trichostatin A, valproic acid, valrubicin, vandetanib,vinblastine, vincristine, vindesine, or vinorelbine.

Methods of treating or preventing a disease with an inflammatorycomponent in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure are also contemplated. The disease may be, for example, lupusor rheumatoid arthritis. The disease may be an inflammatory boweldisease, such as Crohn's disease or ulcerative colitis. The disease withan inflammatory component may be a cardiovascular disease. The diseasewith an inflammatory component may be diabetes, such as type 1 or type 2diabetes. Compounds of the present disclosure may also be used to treatcomplications associated with diabetes. Such complications arewell-known in the art and include, for example, obesity, hypertension,atherosclerosis, coronary heart disease, stroke, peripheral vasculardisease, hypertension, nephropathy, neuropathy, myonecrosis, retinopathyand metabolic syndrome (syndrome X). The disease with an inflammatorycomponent may be a skin disease, such as psoriasis, acne, or atopicdermatitis. Administration of a compound of the present disclosure intreatment methods of such skin diseases may be, for example, topical ororal.

The disease with an inflammatory component may be metabolic syndrome(syndrome X). A patient having this syndrome is characterized as havingthree or more symptoms selected from the following group of fivesymptoms: (1) abdominal obesity; (2) hypertriglyceridemia; (3) lowhigh-density lipoprotein cholesterol (HDL); (4) high blood pressure; and(5) elevated fasting glucose, which may be in the range characteristicof Type 2 diabetes if the patient is also diabetic. Each of thesesymptoms is defined in the Third Report of the National CholesterolEducation Program Expert Panel on Detection, Evaluation and Treatment ofHigh Blood Cholesterol in Adults (Adult Treatment Panel III, or ATPIII), National Institutes of Health, 2001, NIH Publication No. 01-3670,incorporated herein by reference. Patients with metabolic syndrome,whether or not they have or develop overt diabetes mellitus, have anincreased risk of developing the macrovascular and microvascularcomplications that are listed above that occur with type 2 diabetes,such as atherosclerosis and coronary heart disease.

Another general method of the present disclosure entails a method oftreating or preventing a cardiovascular disease in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure. The cardiovascular disease may be,for example, atherosclerosis, cardiomyopathy, congenital heart disease,congestive heart failure, myocarditis, rheumatic heart disease, valvedisease, coronary artery disease, endocarditis, or myocardialinfarction. Combination therapy is also contemplated for such methods.For example, such methods may further comprise administering apharmaceutically effective amount of a second drug. The second drug maybe, for example, a cholesterol lowering drug, an anti-hyperlipidemic, acalcium channel blocker, an anti-hypertensive, or an HMG-CoA reductaseinhibitor. Non-limiting examples of second drugs include amlodipine,aspirin, ezetimibe, felodipine, lacidipine, lercanidipine, nicardipine,nifedipine, nimodipine, nisoldipine or nitrendipine. Other non-limitingexamples of second drugs include atenolol, bucindolol, carvedilol,clonidine, doxazosin, indoramin, labetalol, methyldopa, metoprolol,nadolol, oxprenolol, phenoxybenzamine, phentolamine, pindolol, prazosin,propranolol, terazosin, timolol or tolazoline. The second drug may be,for example, a statin, such as atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin orsimvastatin.

Methods of treating or preventing a neurodegenerative disease in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure are alsocontemplated. The neurodegenerative disease may, for example, beselected from the group consisting of Parkinson's disease, Alzheimer'sdisease, multiple sclerosis (MS), Huntington's disease and amyotrophiclateral sclerosis. In particular embodiments, the neurodegenerativedisease is Alzheimer's disease. In particular embodiments, theneurodegenerative disease is MS, such as primary progressive,relapsing-remitting secondary progressive or progressive relapsing MS.The subject may be, for example, a primate. The subject may be a human.

In particular embodiments of methods of treating or preventing aneurodegenerative disease in a subject, comprising administering to thesubject a pharmaceutically effective amount of a compound of the presentdisclosure, the treatment suppresses the demyelination of neurons in thesubject's brain or spinal cord. In certain embodiments, the treatmentsuppresses inflammatory demyelination. In certain embodiments, thetreatment suppresses the transection of neuron axons in the subject'sbrain or spinal cord. In certain embodiments, the treatment suppressesthe transection of neurites in the subject's brain or spinal cord. Incertain embodiments, the treatment suppresses neuronal apoptosis in thesubject's brain or spinal cord. In certain embodiments, the treatmentstimulates the remyelination of neuron axons in the subject's brain orspinal cord. In certain embodiments, the treatment restores lostfunction after an MS attack. In certain embodiments, the treatmentprevents a new MS attack. In certain embodiments, the treatment preventsa disability resulting from an MS attack.

One general aspect of the present disclosure contemplates a method oftreating or preventing a disorder characterized by overexpression ofiNOS genes in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure.

Another general aspect of the present disclosure contemplates a methodof inhibiting IFN-γ-induced nitric oxide production in cells of asubject, comprising administering to said subject a pharmaceuticallyeffective amount of a compound of the present disclosure.

Yet another general method of the present disclosure contemplates amethod of treating or preventing a disorder characterized byoverexpression of COX-2 genes in a subject, comprising administering tothe subject a pharmaceutically effective amount of compound of thepresent disclosure.

Methods of treating renal/kidney disease (RKD) in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure are also contemplated. See U.S.patent application Ser. No. 12/352,473, which is incorporated byreference herein in its entirety. The RKD may result from, for example,a toxic insult. The toxic insult may result from, for example, animaging agent or a drug. The drug may be a chemotherapeutic, forexample. The RKD may result from ischemia/reperfusion injury, in certainembodiments. In certain embodiments, the RKD results from diabetes orhypertension. The RKD may result from an autoimmune disease. The RKD maybe further defined as chronic RKD, or acute RKD.

In certain methods of treating renal/kidney disease (RKD) in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure, the subject hasundergone or is undergoing dialysis. In certain embodiments, the subjecthas undergone or is a candidate to undergo kidney transplant. Thesubject may be a primate. The primate may be a human. The subject inthis or any other method may be, for example, a cow, horse, dog, cat,pig, mouse, rat or guinea pig.

Also contemplated by the present disclosure is a method for improvingglomerular filtration rate or creatinine clearance in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure.

Methods of synthesis of compounds of the present disclosure are alsocontemplated. For example, certain embodiments contemplate a method ofmaking a first compound, wherein the first compound is as follows:

comprising reacting a compound of the following formula with acid:

Kits are also contemplated by the present disclosure, such as a kitcomprising: a compound of the present disclosure; and instructions whichcomprise one or more forms of information selected from the groupconsisting of indicating a disease state for which the compound is to beadministered, storage information for the compound, dosing informationand instructions regarding how to administer the compound. The kit maycomprise a compound of the present disclosure in a multiple dose form.

In certain embodiments, compounds of the present disclosure may be usedin preventing and treating diseases and disorders whose pathologyinvolves oxidative stress, inflammation, and dysregulation ofinflammatory signaling pathways. In particular embodiments, compounds ofthe invention may be used in treating diseases and disorderscharacterized by overexpression of inducible nitric oxide synthase(iNOS), inducible cyclooxygenase (COX-2), or both, in affected tissues;high levels of production of reactive oxygen species (ROS) or reactivenitrogen species (RNS) such as superoxide, hydrogen peroxide, nitricoxide or peroxynitrite; or excessive production of inflammatorycytokines or other inflammation-related proteins such as TNFα, IL-6,IL-1, IL-8, ICAM-1, VCAM-1, and VEGF. Such diseases or disorders may, insome embodiments, involve undesirable proliferation of certain cells, asin the case of cancer (e.g., solid tumors, leukemias, myelomas,lymphomas, and other cancers), fibrosis associated with organ failure,or excessive scarring. Other such disorders include (but are not limitedto) autoimmune diseases such as lupus, rheumatoid arthritis,juvenile-onset diabetes, multiple sclerosis, psoriasis, and Crohn'sdisease; cardiovascular diseases such as atherosclerosis, heart failure,myocardial infarction, acute coronary syndrome, restenosis followingvascular surgery, hypertension, and vasculitis; neurodegenerative orneuromuscular diseases such as Alzheimer's disease, Parkinson's disease,Huntington's disease, ALS, and muscular dystrophy; neurologicaldisorders such as epilepsy and dystonia; neuropsychiatric conditionssuch as major depression, bipolar disorder, post-traumatic stressdisorder, schizophrenia, anorexia nervosa, ADHD, and autism-spectrumdisorders; retinal diseases such as macular degeneration, diabeticretinopathy, glaucoma, and retinitis; chronic and acute pain syndromes,including inflammatory and neuropathic pain; hearing loss and tinnitus;diabetes and complications of diabetes, including metabolic syndrome,diabetic nephropathy, diabetic neuropathy, and diabetic ulcers;respiratory diseases such as asthma, chronic obstructive pulmonarydisease, acute respiratory distress syndrome, and cystic fibrosis;inflammatory bowel diseases; osteoporosis, osteoarthritis, and otherdegenerative conditions of bone and cartilage; acute or chronic organfailure, including renal failure, liver failure (including cirrhosis andhepatitis), and pancreatitis; ischemia-reperfusion injury associatedwith thrombotic or hemorrhagic stroke, subarachnoid hemorrhage, cerebralvasospasm, myocardial infarction, shock, or trauma; complications oforgan or tissue transplantation including acute or chronic transplantfailure or rejection and graft-versus-host disease; skin diseasesincluding atopic dermatitis and acne; sepsis and septic shock; excessiveinflammation associated with infection, including respiratoryinflammation associated with influenza and upper respiratory infections;mucositis associated with cancer therapy, including radiation therapy orchemotherapy; and severe burns.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula doesn't mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The invention may be better understood by reference to oneof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1-16 and 20-24. Inhibition of NO Production. RAW264.7 macrophageswere pre-treated with DMSO or drugs at various concentrations (μM) for 2hours, then treated with IFNγ for 24 hours. NO concentration in mediawas determined using a Griess reagent system; cell viability wasdetermined using the WST-1 reagent.

FIGS. 17-18. Suppression of iNOS mRNA induction. RAW264.7 mousemacrophages were pre-treated for 2 hours with compounds at the indicatedconcentrations and subsequently stimulated with 10 ng/ml IFNγ for anadditional 2 hours. mRNA levels of iNOS were quantified by qPCR and areshown relative to the vehicle-treated IFNγ-stimulated sample which wasnormalized to a value of 1. Values are averages of duplicate PCRreactions, each with triplicate wells.

FIG. 19. iNOS Western Blot in RAW264.7 mouse macrophages. RAW264.7 cellswere pre-treated for 2 hours with indicated compounds and subsequentlystimulated with 10 ng/ml IFNγ for an additional 24 hours. iNOS proteinlevels were assayed by immunoblotting. Actin was used as a loadingcontrol.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Disclosed herein are, for example, new compounds with antioxidant andanti-inflammatory properties, methods for their manufacture, and methodsfor their use, including for the treatment and/or prevention of disease.

I. DEFINITIONS

“Basic skeleton” is defined as: (1) the atoms and σ-bonds forming thebackbone of each of two rings of an organic compound, e.g., a natural ornon-natural product, that are furthest apart from one another when thenatural product is drawn as a two-dimensional structural formula,provided that the two rings are connected to one another through atleast through one backbone consisting entirely of carbon-carbon bonds(e.g., single, double, triple, aromatic, or non-aromatic); and (2) theatoms and σ-bonds of any backbone connecting the two rings defined in(1).

As used herein, “hydrogen” means —H; “hydroxy” means —OH; “oxo” means═O; “halo” means independently —F, —Cl, —Br or —I; “amino” means —NH₂(see below for definitions of groups containing the term amino, e.g.,alkylamino); “hydroxyamino” means —NHOH; “nitro” means —NO₂; imino means═NH (see below for definitions of groups containing the term imino,e.g., alkylamino); “cyano” means —CN; “azido” means —N₃; “mercapto”means —SH; “thio” means ═S; “sulfonamido” means —NHS(O)₂—(see below fordefinitions of groups containing the term sulfonamido, e.g.,alkylsulfonamido); “sulfonyl” means —S(O)₂—(see below for definitions ofgroups containing the term sulfonyl, e.g., alkylsulfonyl); and “silyl”means —SiH₃ (see below for definitions of group(s) containing the termsilyl, e.g., alkylsilyl).

For the groups below, the following parenthetical subscripts furtherdefine the groups as follows: “(Cn)” defines the exact number (n) ofcarbon atoms in the group. “(C≦n)” defines the maximum number (n) ofcarbon atoms that can be in the group, with the minimum number of carbonatoms in such at least one, but otherwise as small as possible for thegroup in question. E.g., it is understood that the minimum number ofcarbon atoms in the group “alkenyl_((C≦8))” is 2. For example,“alkoxy_((C≦10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3-10 carbon atoms)). (Cn-n′) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group.Similarly, “alkyl_((C2-10))” designates those alkyl groups having from 2to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3-10 carbon atoms)).

The term “alkyl” when used without the “substituted” modifier refers toa non-aromatic monovalent group with a saturated carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (iso-Pr), —CH(CH₂)₂ (cyclopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(iso-butyl), —C(CH₃)₃ (tert-butyl), —CH₂C(CH₃)₃ (neo-pentyl),cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl arenon-limiting examples of alkyl groups. The term “substituted alkyl”refers to a non-aromatic monovalent group with a saturated carbon atomas the point of attachment, a linear or branched, cyclo, cyclic oracyclic structure, no carbon-carbon double or triple bonds, and at leastone atom independently selected from the group consisting of N, O, F,Cl, Br, I, Si, P, and S. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂Cl, —CH₂Br, —CH₂SH, —CF₃,—CH₂CN, —CH₂C(O)H, —CH₂C(O)OH, —CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)NHCH₃,—CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OCH₂CF₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂NHCH₃,—CH₂N(CH₃)₂, —CH₂CH₂Cl, —CH₂CH₂OH, —CH₂CF₃, —CH₂CH₂OC(O)CH₃,—CH₂CH₂NHCO₂C(CH₃)₃, and —CH₂Si(CH₃)₃.

The term “alkanediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkanediyl group isattached with two σ-bonds, with one or two saturated carbon atom(s) asthe point(s) of attachment, a linear or branched, cyclo, cyclic oracyclic structure, no carbon-carbon double or triple bonds, and no atomsother than carbon and hydrogen. The groups, —CH₂-(methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, —CH₂CH₂CH₂—, and

are non-limiting examples of alkanediyl groups. The term “substitutedalkanediyl” refers to a non-aromatic monovalent group, wherein thealkynediyl group is attached with two σ-bonds, with one or two saturatedcarbon atom(s) as the point(s) of attachment, a linear or branched,cyclo, cyclic or acyclic structure, no carbon-carbon double or triplebonds, and at least one atom independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. The following groups arenon-limiting examples of substituted alkanediyl groups: —CH(F)—, —CF₂—,—CH(Cl)—, —CH(OH)—, —CH(OCH₃)—, and —CH₂CH(Cl)—.

The term “alkenyl” when used without the “substituted” modifier refersto a monovalent group with a nonaromatic carbon atom as the point ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples of alkenyl groups include: —CH═CH₂ (vinyl), —CH═CHCH₃,—CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl), —CH₂CH═CHCH₃, and —CH═CH—C₆H₅. Theterm “substituted alkenyl” refers to a monovalent group with anonaromatic carbon atom as the point of attachment, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, alinear or branched, cyclo, cyclic or acyclic structure, and at least oneatom independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, arenon-limiting examples of substituted alkenyl groups.

The term “alkenediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkenediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. The groups, —CH═CH—,—CH═C(CH₃)CH₂—, —CH═CHCH₂—, and

are non-limiting examples of alkenediyl groups. The term “substitutedalkenediyl” refers to a non-aromatic divalent group, wherein thealkenediyl group is attached with two σ-bonds, with two carbon atoms aspoints of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one nonaromatic carbon-carbon double bond, nocarbon-carbon triple bonds, and at least one atom independently selectedfrom the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Thefollowing groups are non-limiting examples of substituted alkenediylgroups: —CF═CH—, —C(OH)═CH—, and —CH₂CH═C(Cl)—.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent group with a nonaromatic carbon atom as the point ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and no atoms other than carbon andhydrogen. The groups, —C≡CH, —C≡CCH₃, —C≡CC₆H₅ and —CH₂C≡CCH₃, arenon-limiting examples of alkynyl groups. The term “substituted alkynyl”refers to a monovalent group with a nonaromatic carbon atom as the pointof attachment and at least one carbon-carbon triple bond, a linear orbranched, cyclo, cyclic or acyclic structure, and at least one atomindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S. The group, —C≡CSi(CH₃)₃, is a non-limiting example of asubstituted alkynyl group.

The term “alkynediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkynediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and no atoms other than carbon andhydrogen. The groups, —C≡C—, —C≡CCH₂—, and —C≡CCH(CH₃)— are non-limitingexamples of alkynediyl groups. The term “substituted alkynediyl” refersto a non-aromatic divalent group, wherein the alkynediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and at least one atom independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.The groups —C≡CCFH— and —C≡CHCH(Cl)— are non-limiting examples ofsubstituted alkynediyl groups.

The term “aryl” when used without the “substituted” modifier refers to amonovalent group with an aromatic carbon atom as the point ofattachment, said carbon atom forming part of a six-membered aromaticring structure wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen.Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl,(dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), —C₆H₄CH₂CH₂CH₃(propylphenyl), —C₆H₄CH(CH₃)₂, —C₆H₄CH(CH₂)₂, —C₆H₃(CH₃)CH₂CH₃(methylethylphenyl), —C₆H₄CH═CH₂ (vinylphenyl), —C₆H₄CH═CHCH₃,—C₆H₄C≡CH, —C₆H₄C≡CCH₃, naphthyl, and the monovalent group derived frombiphenyl. The term “substituted aryl” refers to a monovalent group withan aromatic carbon atom as the point of attachment, said carbon atomforming part of a six-membered aromatic ring structure wherein the ringatoms are all carbon, and wherein the monovalent group further has atleast one atom independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S, Non-limiting examples of substituted arylgroups include the groups: —C₆H₄F, —C₆H₄Cl, —C₆H₄Br, —C₆H₄I, —C₆H₄OH,—C₆H₄OCH₃, —C₆H₄OCH₂CH₃, —C₆H₄OC(O)CH₃, —C₆H₄NH₂, —C₆H₄NHCH₃,—C₆H₄N(CH₃)₂, —C₆H₄CH₂OH, —C₆H₄CH₂OC(O)CH₃, —C₆H₄CH₂NH₂, —C₆H₄CF₃,—C₆H₄CN, —C₆H₄CHO, —C₆H₄CHO, —C₆H₄C(O)CH₃, —C₆H₄C(O)C₆H₅, —C₆H₄CO₂H,—C₆H₄CO₂CH₃, —C₆H₄CONH₂, —C₆H₄CONHCH₃, and —C₆H₄CON(CH₃)₂.

The term “arenediyl” when used without the “substituted” modifier refersto a divalent group, wherein the arenediyl group is attached with twoσ-bonds, with two aromatic carbon atoms as points of attachment, saidcarbon atoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen.Non-limiting examples of arenediyl groups include:

The term “substituted arenediyl” refers to a divalent group, wherein thearenediyl group is attached with two σ-bonds, with two aromatic carbonatoms as points of attachment, said carbon atoms forming part of one ormore six-membered aromatic rings structure(s), wherein the ring atomsare all carbon, and wherein the divalent group further has at least oneatom independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples of aralkyls are: phenylmethyl(benzyl, Bn), 1-phenyl-ethyl, 2-phenyl-ethyl, indenyl and2,3-dihydro-indenyl, provided that indenyl and 2,3-dihydro-indenyl areonly examples of aralkyl in so far as the point of attachment in eachcase is one of the saturated carbon atoms. When the term “aralkyl” isused with the “substituted” modifier, either one or both the alkanediyland the aryl is substituted. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, 2-oxo-2-phenyl-ethyl(phenylcarbonylmethyl), 2-chloro-2-phenyl-ethyl, chromanyl where thepoint of attachment is one of the saturated carbon atoms, andtetrahydroquinolinyl where the point of attachment is one of thesaturated atoms.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent group with an aromatic carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of an aromatic ring structure wherein at least one of thering atoms is nitrogen, oxygen or sulfur, and wherein the monovalentgroup consists of no atoms other than carbon, hydrogen, aromaticnitrogen, aromatic oxygen and aromatic sulfur. Non-limiting examples ofaryl groups include acridinyl, furanyl, imidazoimidazolyl,imidazopyrazolyl, imidazopyridinyl, imidazopyrimidinyl, indolyl,indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl,pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl,tetrahydroquinolinyl, thienyl, triazinyl, pyrrolopyridinyl,pyrrolopyrimidinyl, pyrrolopyrazinyl, pyrrolotriazinyl,pyrroloimidazolyl, chromenyl (where the point of attachment is one ofthe aromatic atoms), and chromanyl (where the point of attachment is oneof the aromatic atoms). The term “substituted heteroaryl” refers to amonovalent group with an aromatic carbon atom or nitrogen atom as thepoint of attachment, said carbon atom or nitrogen atom forming part ofan aromatic ring structure wherein at least one of the ring atoms isnitrogen, oxygen or sulfur, and wherein the monovalent group further hasat least one atom independently selected from the group consisting ofnon-aromatic nitrogen, non-aromatic oxygen, non aromatic sulfur F, Cl,Br, I, Si, and P.

The term “heteroarenediyl” when used without the “substituted” modifierrefers to a divalent group, wherein the heteroarenediyl group isattached with two σ-bonds, with an aromatic carbon atom or nitrogen atomas the point of attachment, said carbon atom or nitrogen atom twoaromatic atoms as points of attachment, said carbon atoms forming partof one or more six-membered aromatic ring structure(s) wherein the ringatoms are all carbon, and wherein the monovalent group consists of noatoms other than carbon and hydrogen. Non-limiting examples ofheteroarenediyl groups include:

The term “substituted heteroarenediyl” refers to a divalent group,wherein the heteroarenediyl group is attached with two σ-bonds, with twoaromatic carbon atoms as points of attachment, said carbon atoms formingpart of one or more six-membered aromatic rings structure(s), whereinthe ring atoms are all carbon, and wherein the divalent group furtherhas at least one atom independently selected from the group consistingof N, O, F, Cl, Br, I, Si, P, and S.

The term “heteroaralkyl” when used without the “substituted” modifierrefers to the monovalent group -alkanediyl-heteroaryl, in which theterms alkanediyl and heteroaryl are each used in a manner consistentwith the definitions provided above. Non-limiting examples of aralkylsare: pyridylmethyl, and thienylmethyl. When the term “heteroaralkyl” isused with the “substituted” modifier, either one or both the alkanediyland the heteroaryl is substituted.

The term “acyl” when used without the “substituted” modifier refers to amonovalent group with a carbon atom of a carbonyl group as the point ofattachment, further having a linear or branched, cyclo, cyclic oracyclic structure, further having no additional atoms that are notcarbon or hydrogen, beyond the oxygen atom of the carbonyl group. Thegroups, —CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃,—C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)C₆H₄CH₂CH₃,—COC₆H₃(CH₃)₂, and —C(O)CH₂C₆H₅, are non-limiting examples of acylgroups. The term “acyl” therefore encompasses, but is not limited togroups sometimes referred to as “alkyl carbonyl” and “aryl carbonyl”groups. The term “substituted acyl” refers to a monovalent group with acarbon atom of a carbonyl group as the point of attachment, furtherhaving a linear or branched, cyclo, cyclic or acyclic structure, furtherhaving at least one atom, in addition to the oxygen of the carbonylgroup, independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃(methylcarboxyl), —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃, —CO₂C₆H₅, —CO₂CH(CH₃)₂,—CO₂CH(CH₂)₂, —C(O)NH₂ (carbamoyl), —C(O)NHCH₃, —C(O)NHCH₂CH₃,—CONHCH(CH₃)₂, —CONHCH(CH₂)₂, —CON(CH₃)₂, —CONHCH₂CF₃, —CO-pyridyl,—CO-imidazoyl, and —C(O)N₃, are non-limiting examples of substitutedacyl groups. The term “substituted acyl” encompasses, but is not limitedto, “heteroaryl carbonyl” groups.

The term “alkylidene” when used without the “substituted” modifierrefers to the divalent group ═CRR′, wherein the alkylidene group isattached with one σ-bond and one π-bond, in which R and R′ areindependently hydrogen, alkyl, or R and R′ are taken together torepresent alkanediyl. Non-limiting examples of alkylidene groupsinclude: ═CH₂, ═CH(CH₂CH₃), and ═C(CH₃)₂. The term “substitutedalkylidene” refers to the group ═CRR′, wherein the alkylidene group isattached with one σ-bond and one π-bond, in which R and R′ areindependently hydrogen, alkyl, substituted alkyl, or R and R′ are takentogether to represent a substituted alkanediyl, provided that either oneof R and R′ is a substituted alkyl or R and R′ are taken together torepresent a substituted alkanediyl.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃, —OCH₂CH₃,—OCH₂CH₂CH₃, —OCH(CH₃)₂, —OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl.The term “substituted alkoxy” refers to the group —OR, in which R is asubstituted alkyl, as that term is defined above. For example, —OCH₂CF₃is a substituted alkoxy group.

Similarly, the terms “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”,“heteroaryloxy”, “heteroaralkoxy” and “acyloxy”, when used without the“substituted” modifier, refers to groups, defined as —OR, in which R isalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively, as those terms are defined above. When any of the termsalkenyloxy, alkynyloxy, aryloxy, aralkyloxy and acyloxy is modified by“substituted,” it refers to the group —OR, in which R is substitutedalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃, —NHCH₂CH₃, —NHCH₂CH₂CH₃, —NHCH(CH₃)₂, —NHCH(CH₂)₂,—NHCH₂CH₂CH₂CH₃, —NHCH(CH₃)CH₂CH₃, —NHCH₂CH(CH₃)₂, —NHC(CH₃)₃,—NH-cyclopentyl, and —NH-cyclohexyl. The term “substituted alkylamino”refers to the group —NHR, in which R is a substituted alkyl, as thatterm is defined above. For example, —NHCH₂CF₃ is a substitutedalkylamino group.

The term “dialkylamino” when used without the “substituted” modifierrefers to the group —NRR′, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl having two or more saturated carbon atoms, at least two ofwhich are attached to the nitrogen atom. Non-limiting examples ofdialkylamino groups include: —NHC(CH₃)₃, —N(CH₃)CH₂CH₃, —N(CH₂CH₃)₂,N-pyrrolidinyl, and N-piperidinyl. The term “substituted dialkylamino”refers to the group —NRR′, in which R and R′ can be the same ordifferent substituted alkyl groups, one of R or R′ is an alkyl and theother is a substituted alkyl, or R and R′ can be taken together torepresent a substituted alkanediyl with two or more saturated carbonatoms, at least two of which are attached to the nitrogen atom.

The terms “alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, “heteroaralkylamino”, and“alkylsulfonylamino” when used without the “substituted” modifier,refers to groups, defined as —NHR, in which R is alkoxy, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,respectively, as those terms are defined above. A non-limiting exampleof an arylamino group is —NHC₆H₅. When any of the terms alkoxyamino,alkenylamino, alkynylamino, arylamino, aralkylamino, heteroarylamino,heteroaralkylamino and alkylsulfonylamino is modified by “substituted,”it refers to the group —NHR, in which R is substituted alkoxy, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,respectively.

The term “amido” (acylamino), when used without the “substituted”modifier, refers to the group —NHR, in which R is acyl, as that term isdefined above. A non-limiting example of an acylamino group is—NHC(O)CH₃. When the term amido is used with the “substituted” modifier,it refers to groups, defined as —NHR, in which R is substituted acyl, asthat term is defined above. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

The term “alkylimino” when used without the “substituted” modifierrefers to the group ═NR, wherein the alkylimino group is attached withone σ-bond and one π-bond, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylimino groups include:═NCH₃, ═NCH₂CH₃ and ═N-cyclohexyl. The term “substituted alkylimino”refers to the group ═NR, wherein the alkylimino group is attached withone σ-bond and one π-bond, in which R is a substituted alkyl, as thatterm is defined above. For example, ═NCH₂CF₃ is a substituted alkyliminogroup.

Similarly, the terms “alkenylimino”, “alkynylimino”, “arylimino”,“aralkylimino”, “heteroarylimino”, “heteroaralkylimino” and “acylimino”,when used without the “substituted” modifier, refers to groups, definedas ═NR, wherein the alkylimino group is attached with one σ-bond and oneπ-bond, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,heteroaralkyl and acyl, respectively, as those terms are defined above.When any of the terms alkenylimino, alkynylimino, arylimino,aralkylimino and acylimino is modified by “substituted,” it refers tothe group ═NR, wherein the alkylimino group is attached with one σ-bondand one π-bond, in which R is substituted alkenyl, alkynyl, aryl,aralkyl, heteroaryl, heteroaralkyl and acyl, respectively.

The term “fluoroalkyl” when used without the “substituted” modifierrefers to an alkyl, as that term is defined above, in which one or morefluorines have been substituted for hydrogens. The groups, —CH₂F, —CF₃,and —CH₂CF₃ are non-limiting examples of fluoroalkyl groups. The term“substituted fluoroalkyl” refers to a non-aromatic monovalent group witha saturated carbon atom as the point of attachment, a linear orbranched, cyclo, cyclic or acyclic structure, at least one fluorineatom, no carbon-carbon double or triple bonds, and at least one atomindependently selected from the group consisting of N, O, Cl, Br, I, Si,P, and S. The following group is a non-limiting example of a substitutedfluoroalkyl: —CFHOH.

The term “alkylthio” when used without the “substituted” modifier refersto the group —SR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkylthio groups include: —SCH₃, —SCH₂CH₃,—SCH₂CH₂CH₃, —SCH(CH₃)₂, —SCH(CH₂)₂, —S-cyclopentyl, and —S-cyclohexyl.The term “substituted alkylthio” refers to the group —SR, in which R isa substituted alkyl, as that term is defined above. For example,—SCH₂CF₃ is a substituted alkylthio group.

Similarly, the terms “alkenylthio”, “alkynylthio”, “arylthio”,“aralkylthio”, “heteroarylthio”, “heteroaralkylthio”, and “acylthio”,when used without the “substituted” modifier, refers to groups, definedas —SR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,heteroaralkyl and acyl, respectively, as those terms are defined above.When any of the terms alkenylthio, alkynylthio, arylthio, aralkylthio,heteroarylthio, heteroaralkylthio, and acylthio is modified by“substituted,” it refers to the group —SR, in which R is substitutedalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively.

The term “thioacyl” when used without the “substituted” modifier refersto a monovalent group with a carbon atom of a thiocarbonyl group as thepoint of attachment, further having a linear or branched, cyclo, cyclicor acyclic structure, further having no additional atoms that are notcarbon or hydrogen, beyond the sulfur atom of the carbonyl group. Thegroups, —CHS, —C(S)CH₃, —C(S)CH₂CH₃, —C(S)CH₂CH₂CH₃, —C(S)CH(CH₃)₂,—C(S)CH(CH₂)₂, —C(S)C₆H₅, —C(S)C₆H₄CH₃, —C(S)C₆H₄CH₂CH₃,—C(S)C₆H₃(CH₃)₂, and —C(S)CH₂C₆H₅, are non-limiting examples of thioacylgroups. The term “thioacyl” therefore encompasses, but is not limitedto, groups sometimes referred to as “alkyl thiocarbonyl” and “arylthiocarbonyl” groups. The term “substituted thioacyl” refers to aradical with a carbon atom as the point of attachment, the carbon atombeing part of a thiocarbonyl group, further having a linear or branched,cyclo, cyclic or acyclic structure, further having at least one atom, inaddition to the sulfur atom of the carbonyl group, independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.The groups, —C(S)CH₂CF₃, —C(S)O₂H, —C(S)OCH₃, —C(S)OCH₂CH₃,—C(S)OCH₂CH₂CH₃, —C(S)OC₆H₅, —C(S)OCH(CH₃)₂, —C(S)OCH(CH₂)₂, —C(S)NH₂,and —C(S)NHCH₃, are non-limiting examples of substituted thioacylgroups. The term “substituted thioacyl” encompasses, but is not limitedto, “heteroaryl thiocarbonyl” groups.

The term “alkylsulfonyl” when used without the “substituted” modifierrefers to the group —S(O)₂R, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylsulfonyl groups include:—S(O)₂CH₃, —S(O)₂CH₂CH₃, —S(O)₂CH₂CH₂CH₃, —S(O)₂CH(CH₃)₂,—S(O)₂CH(CH₂)₂, —S(O)₂ cyclopentyl, and —S(O)₂-cyclohexyl. The term“substituted alkylsulfonyl” refers to the group —S(O)₂R, in which R is asubstituted alkyl, as that term is defined above. For example,—S(O)₂CH₂CF₃ is a substituted alkylsulfonyl group.

Similarly, the terms “alkenylsulfonyl”, “alkynylsulfonyl”,“arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl”, and“heteroaralkylsulfonyl” when used without the “substituted” modifier,refers to groups, defined as —S(O)₂R, in which R is alkenyl, alkynyl,aryl, aralkyl, heteroaryl, and heteroaralkyl, respectively, as thoseterms are defined above. When any of the terms alkenylsulfonyl,alkynylsulfonyl, arylsulfonyl, aralkylsulfonyl, heteroarylsulfonyl, andheteroaralkylsulfonyl is modified by “substituted,” it refers to thegroup —S(O)₂R, in which R is substituted alkenyl, alkynyl, aryl,aralkyl, heteroaryl and heteroaralkyl, respectively.

The term “alkylammonium” when used without the “substituted” modifierrefers to a group, defined as —NH₂R⁺, —NHRR′⁺, or —NRR′R″⁺, in which R,R′ and R″ are the same or different alkyl groups, or any combination oftwo of R, R′ and R″ can be taken together to represent an alkanediyl.Non-limiting examples of alkylammonium cation groups include:—NH₂(CH₃)⁺, —NH₂(CH₂CH₃)+, —NH₂(CH₂CH₂CH₃)+, —NH(CH₃)₂ ⁺, —NH(CH₂CH₃)₂⁺, —NH(CH₂CH₂CH₃)₂ ⁺, —N(CH₃)₃ ⁺, —N(CH₃)(CH₂CH₃)₂ ⁺, —N(CH₃)₂(CH₂CH₃)⁺,—NH₂C(CH₃)₃ ⁺, —NH(cyclopentyl)₂ ⁺, and —NH₂(cyclohexyl)⁺. The term“substituted alkylammonium” refers —NH₂R⁺, —NHRR′⁺, or —NRR′R″⁺, inwhich at least one of R, R′ and R″ is a substituted alkyl or two of R,R′ and R″ can be taken together to represent a substituted alkanediyl.When more than one of R, R′ and R″ is a substituted alkyl, they can bethe same of different. Any of R, R′ and R″ that are not eithersubstituted alkyl or substituted alkanediyl, can be either alkyl, eitherthe same or different, or can be taken together to represent aalkanediyl with two or more carbon atoms, at least two of which areattached to the nitrogen atom shown in the formula.

The term “alkylsulfonium” when used without the “substituted” modifierrefers to the group —SRR′⁺, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl. Non-limiting examples of alkylsulfonium groups include:—SH(CH₃)⁺, —SH(CH₂CH₃)⁺, —SH(CH₂CH₂CH₃)⁺, —S(CH₃)₂ ⁺, —S(CH₂CH₃)₂ ⁺,—S(CH₂CH₂CH₃)₂ ⁺, —SH(cyclopentyl)⁺, and —SH(cyclohexyl)⁺. The term“substituted alkylsulfonium” refers to the group —SRR′⁺, in which R andR′ can be the same or different substituted alkyl groups, one of R or R′is an alkyl and the other is a substituted alkyl, or R and R′ can betaken together to represent a substituted alkanediyl. For example,—SH(CH₂CF₃)⁺ is a substituted alkylsulfonium group.

The term “alkylsilyl” when used without the “substituted” modifierrefers to a monovalent group, defined as —SiH₂R, —SiHRR′, or —SiRR′R″,in which R, R′ and R″ can be the same or different alkyl groups, or anycombination of two of R, R′ and R″ can be taken together to represent analkanediyl. The groups, —SiH₂CH₃, —SiH(CH₃)₂, —Si(CH₃)₃ and—Si(CH₃)₂C(CH₃)₃, are non-limiting examples of unsubstituted alkylsilylgroups. The term “substituted alkylsilyl” refers —SiH₂R, —SiHRR′, or—SiRR′R″, in which at least one of R, R′ and R″ is a substituted alkylor two of R, R′ and R″ can be taken together to represent a substitutedalkanediyl. When more than one of R, R′ and R″ is a substituted alkyl,they can be the same of different. Any of R, R′ and R″ that are noteither substituted alkyl or substituted alkanediyl, can be either alkyl,either the same or different, or can be taken together to represent aalkanediyl with two or more saturated carbon atoms, at least two ofwhich are attached to the silicon atom.

In addition, atoms making up the compounds of the present disclosure areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C. Similarly, it is contemplated that one or morecarbon atom(s) of a compound of the present disclosure may be replacedby a silicon atom(s). Furthermore, it is contemplated that one or moreoxygen atom(s) of a compound of the present disclosure may be replacedby a sulfur or selenium atom(s).

A compound having a formula that is represented with a dashed bond isintended to include the formulae optionally having zero, one or moredouble bonds. Thus, for example, the structure

includes the structures

and

As will be understood by a person of skill in the art, no one such ringatom forms part of more than one double bond.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

A ring structure shown with an unconnected “R” group, indicates that anyimplicit hydrogen atom on that ring can be replaced with that R group.In the case of a divalent R group (e.g., oxo, imino, thio, alkylidene,etc.), any pair of implicit hydrogen atoms attached to one atom of thatring can be replaced by that R group. This concept is as exemplifiedbelow:

-   -   represents

As used herein, a “chiral auxiliary” refers to a removable chiral groupthat is capable of influencing the stereoselectivity of a reaction.Persons of skill in the art are familiar with such compounds, and manyare commercially available.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent disclosure which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical SaltsProperties, and Use (2002).

As used herein, “predominantly one enantiomer” means that a compoundcontains at least about 85% of one enantiomer, or more preferably atleast about 90% of one enantiomer, or even more preferably at leastabout 95% of one enantiomer, or most preferably at least about 99% ofone enantiomer. Similarly, the phrase “substantially free from otheroptical isomers” means that the composition contains at most about 15%of another enantiomer or diastereomer, more preferably at most about 10%of another enantiomer or diastereomer, even more preferably at mostabout 5% of another enantiomer or diastereomer, and most preferably atmost about 1% of another enantiomer or diastereomer.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present disclosure. The prodrugitself may or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methane-sulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexyl-sulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

The term “saturated” when referring to an atom means that the atom isconnected to other atoms only by means of single bonds.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers.

The invention contemplates that for any stereocenter or axis ofchirality for which stereochemistry has not been defined, thatstereocenter or axis of chirality can be present in its R form, S form,or as a mixture of the R and S forms, including racemic and non-racemicmixtures.

“Substituent convertible to hydrogen in vivo” means any group that isconvertible to a hydrogen atom by enzymological or chemical meansincluding, but not limited to, hydrolysis and hydrogenolysis. Examplesinclude acyl groups, groups having an oxycarbonyl group, amino acidresidues, peptide residues, o-nitrophenylsulfenyl, trimethylsilyl,tetrahydro-pyranyl, diphenylphosphinyl, hydroxy or alkoxy substituentson imino groups, and the like. Examples of acyl groups include formyl,acetyl, trifluoroacetyl, and the like. Examples of groups having anoxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl(—C(O)OC(CH₃)₃), benzyloxycarbonyl, p-methoxy-benzyloxycarbonyl,vinyloxycarbonyl, β-(p-toluenesulfonyl)ethoxycarbonyl, and the like.Suitable amino acid residues include, but are not limited to, residuesof Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp(aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine),Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe(phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp(tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse(homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn(ornithine) and β-Ala. Examples of suitable amino acid residues alsoinclude amino acid residues that are protected with a protecting group.Examples of suitable protecting groups include those typically employedin peptide synthesis, including acyl groups (such as formyl and acetyl),arylmethyloxycarbonyl groups (such as benzyloxycarbonyl andp-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (—C(O)OC(CH₃)₃),and the like. Suitable peptide residues include peptide residuescomprising two to five, and optionally amino acid residues. The residuesof these amino acids or peptides can be present in stereochemicalconfigurations of the D-form, the L-form or mixtures thereof. Inaddition, the amino acid or peptide residue may have an asymmetriccarbon atom. Examples of suitable amino acid residues having anasymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val,Met, Ser, Lys, Thr and Tyr. Peptide residues having an asymmetric carbonatom include peptide residues having one or more constituent amino acidresidues having an asymmetric carbon atom. Examples of suitable aminoacid protecting groups include those typically employed in peptidesynthesis, including acyl groups (such as formyl and acetyl),arylmethyloxycarbonyl groups (such as benzyloxycarbonyl andp-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (—C(O)OC(CH₃)₃),and the like. Other examples of substituents “convertible to hydrogen invivo” include reductively eliminable hydrogenolyzable groups. Examplesof suitable reductively eliminable hydrogenolyzable groups include, butare not limited to, arylsulfonyl groups (such as o-toluenesulfonyl);methyl groups substituted with phenyl or benzyloxy (such as benzyl,trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such asbenzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); andhaloethoxycarbonyl groups (such as β,β,β-trichloroethoxycarbonyl andβ-iodoethoxycarbonyl).

“Therapeutically effective amount” or “pharmaceutically effectiveamount” means that amount which, when administered to a subject orpatient for treating a disease, is sufficient to effect such treatmentfor the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

As used herein, the term “water soluble” means that the compounddissolves in water at least to the extent of 0.010 mole/liter or isclassified as soluble according to literature precedence.

Other abbreviations used herein are as follows: DMSO, dimethylsulfoxide; NO, nitric oxide; iNOS, inducible nitric oxide synthase;COX-2, cyclooxygenase-2; NGF, nerve growth factor; IBMX,isobutylmethylxanthine; FBS, fetal bovine serum; GPDH, glycerol3-phosphate dehydrogenase; RXR, retinoid X receptor; TGF-β, transforminggrowth factor-β; IFNγ or IFN-γ, interferon-γ; LPS, bacterial endotoxiclipopolysaccharide; TNFα or TNF-α, tumor necrosis factor-α; IL-1β,interleukin-1β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; TCA,trichloroacetic acid; HO-1, inducible heme oxygenase.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite.

Rather, all terms used are believed to describe the invention in termssuch that one of ordinary skill can appreciate the scope and practicethe present disclosure.

II. SYNTHETIC METHODS

One of the anti-inflammatory pharmacores of the present invention may berepresented by the following moiety:

wherein X, R₁ and R₂ are as defined above and in the claim below. Thispharmacore is found in a variety of synthetic triterpenoids, such asthose described by Honda et al. (2000a); Honda et al., (2000b); Honda etal. (2002); and U.S. application Ser. No. 11/941,820, each of which isincorporated herein by reference. Other compounds comprising such apharmacore are described in applications filed concurrently with thepresent application, each of which is incorporated herein by reference:U.S. patent application by Eric Anderson, Xin Jiang, Xiaofeng Liu;Melean Visnick, entitled “Antioxidant Inflammation Modulators: OleanolicAcid Derivatives With Saturation in the C-Ring,” filed Apr. 20, 2009;U.S. patent application by Eric Anderson, Xin Jiang and Melean Visnick,entitled “Antioxidant Inflammation Modulators: Oleanolic AcidDerivatives with Amino and Other Modifications At C-17,” filed Apr. 20,2009; U.S. patent application by Xin Jiang, Jack Greiner, Lester L.Maravetz, Stephen S. Szucs, Melean Visnick, entitled “AntioxidantInflammation Modulators: Novel Derivatives of Oleanolic Acid,” filedApr. 20, 2009; and U.S. patent application by Xin Jiang, Xioafeng Liu,Jack Greiner, Stephen S. Szucs, Melean Visnick entitled, “AntioxidantInflammation Modulators: C-17 Homologated Oleanolic Acid Derivatives,”filed Apr. 20, 2009. Many of these compounds (“the referencedcompounds”) have shown a variety of anti-inflammatory-related activitiesincluding, for example, anti-proliferative activities and/or antioxidantactivities such as induction of heme oxygenase-1 (HO-1) in vitro and invivo, induction of CD11b expression, inhibition of iNOS induction,inhibition of COX-2 induction, inhibition of NO production, induction ofapoptosis in cancer cells, inhibition of NF-κB, activation of the JNKpathway, and phase 2 induction (elevation of NAD(P)H-quinoneoxidoreductase and HO-1). Induction of the Phase 2 response is relatedto activation of the transcription factor Nrf2, which has been shown toactivate the antioxidant response element (ARE) in the promoter regionof many antioxidant, anti-inflammatory, and cytoprotective genes, andPhase 2 activation is highly correlated with potent inhibition of NOproduction in activated macrophages (e.g., Dinkova-Kostova et al., Proc.Natl. Acad. Sci. USA. 2005; 102(12):4584-9). As (i) compounds of thepresent invention share a common pharmacore with many of the referencedcompounds, and (ii) like the referenced compounds, compounds of thepresent invention have also been shown to inhibit NO production,compounds of the present invention likely also exhibit one or more ofthe anti-inflammatory activities displayed by the referenced compounds.As shown herein, introduction of this pharmacore into molecules showinglow or modest potency as inhibitors of NO production consistentlyprovides compounds with significantly enhanced potency. For example,compound C0009 has an IC₅₀ of 100 nM in the NO assay, vs. the IC₅₀ of1.5 micromolar for the parent compound, curcumin. Similarly, DHEA wasinactive in the NO assay (IC₅₀>10 micromolar), vs. the IC₅₀ of 130 nMfor compound D0018. Preferably, the modified compound would have an IC₅₀less than 1.25 micromolar. Still more preferably, the modified compoundwould have an IC₅₀ less than 500 nM. In certain embodiments, compoundsamenable to the modifications contemplated herein include, but are notlimited to, triterpenoids (non-limiting examples include glycyrrhetinicacid, boswellic acid, faradiol, calenduladiol, and moronic acid),saponins (e.g., ginsenoside), avicins, resveratrol, curcumin, gossypol,epigallocatechin, epigallocatechin-3-gallate (EGCG), gossypol, lapachol,other flavonoids (non-limiting examples include quercetin, daidzein,luteolin, coumarin, wogonin and baicalin), dehydroandrosterone (DHEA),cholic acid, deoxycholic acid, ginsenoside (e.g., 20(S)-ginsenoside),silymarin, anthocyanins, avenanthramides, cucurbitacins, aloesin,aloe-emodin, and/or tubeimosides.

Non-limiting examples of compounds of the present disclosure whichcontain this particular pharmacore include:

The compounds of the present disclosure were made using the methodsoutlined below and in the Examples section (Example 2 and 3). Thesemethods can be further modified and optimized using the principles andtechniques of organic chemistry as applied by a person skilled in theart. Such principles and techniques are taught, for example, in March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007),which is incorporated by reference herein.

III. BIOLOGICAL ACTIVITY

Compounds of the present disclosure have been tested for inhibition ofNO production. The results of these experiments are shown in the figuresand in Table 1, below. Experimental details are provided in Example 1.

TABLE 1 Suppression of IFNγ-induced NO production. RAW264.7 (20 ng/mlIFNγ) Working ID MW NO IC₅₀ WST-1 IC₅₀ Curcumin 368.38 1.35 μM 4 μMC0008 593.83 0.34 μM 5 μM C0009 435.30 0.1 μM 1 μM C0010 479.23 0.7 μM 5μM Resveratrol 456.70 >10 μM >10 μM R00141 253.30 3 μM >10 μM R00142281.35 1.2 μM >10 μM R00142-1 267.32 1.6 μM >10 μM DHEA 288.42 >10μM >10 μM D0014 357.49 2.4 μM >10 μM D0015 313.20 2.9 μM >10 μM D0016413.67 0.15 μM 10 μM D0017 341.49 0.14 μM 10 μM D0018 339.47 0.13 μM >10μM Hecogenin 472.66 >10 μM >10 μM H0001 449.26 0.15 μM 2.5 μM 63290416.56 ~3 μM ~10 μM 63291 460.61 ~1.5 μM >10 μM 63304 405.53 ~30 nM ~400nM 63311 361.48 ~20 nM ~0.75 μM 63313 393.57 ~0.45 μM ~2.5 μM 63317407.55 ~70 nM See FIG. 22 63318 363.49 ~50 nM See FIG. 23 63312 531.68~3.5 μM >200 nM 63314 443.59 ~8 μM >200 nM 63329 365.51 ~60 nM See FIG.24

IV. DISEASES ASSOCIATED WITH INFLAMMATION AND/OR OXIDATIVE STRESS

Inflammation is a biological process that provides resistance toinfectious or parasitic organisms and the repair of damaged tissue.Inflammation is commonly characterized by localized vasodilation,redness, swelling, and pain, the recruitment of leukocytes to the siteof infection or injury, production of inflammatory cytokines such asTNF-α and IL-1, and production of reactive oxygen or nitrogen speciessuch as hydrogen peroxide, superoxide and peroxynitrite. In later stagesof inflammation, tissue remodeling, angiogenesis, and scar formation(fibrosis) may occur as part of the wound healing process. Under normalcircumstances, the inflammatory response is regulated and temporary andis resolved in an orchestrated fashion once the infection or injury hasbeen dealt with adequately. However, acute inflammation can becomeexcessive and life-threatening if regulatory mechanisms fail.Alternatively, inflammation can become chronic and cause cumulativetissue damage or systemic complications.

Many serious and intractable human diseases involve dysregulation ofinflammatory processes, including diseases such as cancer,atherosclerosis, and diabetes, which were not traditionally viewed asinflammatory conditions. In the case of cancer, the inflammatoryprocesses are associated with tumor formation, progression, metastasis,and resistance to therapy. Atherosclerosis, long viewed as a disorder oflipid metabolism, is now understood to be primarily an inflammatorycondition, with activated macrophages playing an important role in theformation and eventual rupture of atherosclerotic plaques. Activation ofinflammatory signaling pathways has also been shown to play a role inthe development of insulin resistance, as well as in the peripheraltissue damage associated with diabetic hyperglycemia. Excessiveproduction of reactive oxygen species and reactive nitrogen species suchas superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite is ahallmark of inflammatory conditions. Evidence of dysregulatedperoxynitrite production has been reported in a wide variety of diseases(Szabo et al., 2007; Schulz et al., 2008; Forstermann, 2006; Pall,2007).

Autoimmune diseases such as rheumatoid arthritis, lupus, psoriasis, andmultiple sclerosis involve inappropriate and chronic activation ofinflammatory processes in affected tissues, arising from dysfunction ofself vs. non-self recognition and response mechanisms in the immunesystem. In neurodegenerative diseases such as Alzheimer's andParkinson's diseases, neural damage is correlated with activation ofmicroglia and elevated levels of pro-inflammatory proteins such asinducible nitric oxide synthase (iNOS). Chronic organ failure such asrenal failure, heart failure, and chronic obstructive pulmonary diseaseis closely associated with the presence of chronic oxidative stress andinflammation, leading to the development of fibrosis and eventual lossof organ function.

Many other disorders involve oxidative stress and inflammation inaffected tissues, including inflammatory bowel disease; inflammatoryskin diseases; mucositis related to radiation therapy and chemotherapy;eye diseases such as uveitis, glaucoma, macular degeneration, andvarious forms of retinopathy; transplant failure and rejection;ischemia-reperfusion injury; chronic pain; degenerative conditions ofthe bones and joints including osteoarthritis and osteoporosis; asthmaand cystic fibrosis; seizure disorders; and neuropsychiatric conditionsincluding schizophrenia, depression, bipolar disorder, post-traumaticstress disorder, attention deficit disorders, autism-spectrum disorders,and eating disorders such as anorexia nervosa. Dysregulation ofinflammatory signaling pathways is believed to be a major factor in thepathology of muscle wasting diseases including muscular dystrophy andvarious forms of cachexia.

A variety of life-threatening acute disorders also involve dysregulatedinflammatory signaling, including acute organ failure involving thepancreas, kidneys, liver, or lungs, myocardial infarction or acutecoronary syndrome, stroke, septic shock, trauma, severe burns, andanaphylaxis.

Many complications of infectious diseases also involve dysregulation ofinflammatory responses. Although an inflammatory response can killinvading pathogens, an excessive inflammatory response can also be quitedestructive and in some cases can be a primary source of damage ininfected tissues. Furthermore, an excessive inflammatory response canalso lead to systemic complications due to overproduction ofinflammatory cytokines such as TNF-α and IL-1. This is believed to be afactor in mortality arising from severe influenza, severe acuterespiratory syndrome, and sepsis.

The aberrant or excessive expression of either iNOS or cyclooxygenase-2(COX-2) has been implicated in the pathogenesis of many diseaseprocesses. For example, it is clear that NO is a potent mutagen (Tamirand Tannebaum, 1996), and that nitric oxide can also activate COX-2(Salvemini et al., 1994). Furthermore, there is a marked increase iniNOS in rat colon tumors induced by the carcinogen, azoxymethane(Takahashi et al., 1997). A series of synthetic triterpenoid analogs ofoleanolic acid have been shown to be powerful inhibitors of cellularinflammatory processes, such as the induction by IFN-γ of induciblenitric oxide synthase (iNOS) and of COX-2 in mouse macrophages. SeeHonda et al. (2000a); Honda et al. (2000b), and Honda et al. (2002),which are all incorporated herein by reference.

In one aspect, compounds of the invention are characterized by theirability to inhibit the production of nitric oxide in macrophage-derivedRAW 264.7 cells induced by exposure to γ-interferon. They are furthercharacterized by their ability to induce the expression of antioxidantproteins such as NQO1 and reduce the expression of pro-inflammatoryproteins such as COX-2 and inducible nitric oxide synthase (iNOS). Theseproperties are relevant to the treatment of a wide array of diseasesinvolving oxidative stress and dysregulation of inflammatory processesincluding cancer, mucositis resulting from radiation therapy orchemotherapy, autoimmune diseases, cardiovascular diseases includingatherosclerosis, ischemia-reperfusion injury, acute and chronic organfailure including renal failure and heart failure, respiratory diseases,diabetes and complications of diabetes, severe allergies, transplantrejection, graft-versus-host disease, neurodegenerative diseases,diseases of the eye and retina, acute and chronic pain, degenerativebone diseases including osteoarthritis and osteoporosis, inflammatorybowel diseases, dermatitis and other skin diseases, sepsis, burns,seizure disorders, and neuropsychiatric disorders.

Without being bound by theory, the activation of theantioxidant/anti-inflammatory Keap1/Nrf2/ARE pathway is believed to beimplicated in both the anti-inflammatory and anti-carcinogenicproperties of the present oleanolic acid derivatives.

In another aspect, compounds of the invention may be used for treating asubject having a condition caused by elevated levels of oxidative stressin one or more tissues. Oxidative stress results from abnormally high orprolonged levels of reactive oxygen species such as superoxide, hydrogenperoxide, nitric oxide, and peroxynitrite (formed by the reaction ofnitric oxide and superoxide). The oxidative stress may be accompanied byeither acute or chronic inflammation. The oxidative stress may be causedby mitochondrial dysfunction, by activation of immune cells such asmacrophages and neutrophils, by acute exposure to an external agent suchas ionizing radiation or a cytotoxic chemotherapy agent (e.g.,doxorubicin), by trauma or other acute tissue injury, byischemia/reperfusion, by poor circulation or anemia, by localized orsystemic hypoxia or hyperoxia, by elevated levels of inflammatorycytokines and other inflammation-related proteins, and/or by otherabnormal physiological states such as hyperglycemia or hypoglycemia.

In animal models of many such conditions, stimulating expression ofinducible heme oxygenase (HO-1), a target gene of the Nrf2 pathway, hasbeen shown to have a significant therapeutic effect including models ofmyocardial infarction, renal failure, transplant failure and rejection,stroke, cardiovascular disease, and autoimmune disease (e.g., Sacerdotiet al., 2005; Abraham & Kappas, 2005; Bach, 2006; Araujo et al., 2003;Liu et al., 2006; Ishikawa et al., 2001; Kruger et al., 2006; Satoh etal., 2006; Zhou et al., 2005; Morse and Choi, 2005; Morse and Choi,2002). This enzyme breaks free heme down into iron, carbon monoxide(CO), and biliverdin (which is subsequently converted to the potentantioxidant molecule, bilirubin).

In another aspect, compounds of this invention may be used in preventingor treating tissue damage or organ failure, acute and chronic, resultingfrom oxidative stress exacerbated by inflammation. Examples of diseasesthat fall in this category include: heart failure, liver failure,transplant failure and rejection, renal failure, pancreatitis, fibroticlung diseases (cystic fibrosis and COPD, among others), diabetes(including complications), atherosclerosis, ischemia-reperfusion injury,glaucoma, stroke, autoimmune disease, autism, macular degeneration, andmuscular dystrophy. For example, in the case of autism, studies suggestthat increased oxidative stress in the central nervous system maycontribute to the development of the disease (Chauhan and Chauhan,2006).

Evidence also links oxidative stress and inflammation to the developmentand pathology of many other disorders of the central nervous system,including psychiatric disorders such as psychosis, major depression, andbipolar disorder; seizure disorders such as epilepsy; pain and sensorysyndromes such as migraine, neuropathic pain or tinnitus; and behavioralsyndromes such as the attention deficit disorders. See, e.g., Dickersonet al., 2007; Hanson et al., 2005; Kendall-Tackett, 2007; Lencz et al.,2007; Dudhgaonkar et al., 2006; Lee et al., 2007; Morris et al., 2002;Ruster et al., 2005; McIver et al., 2005; Sarchielli et al., 2006;Kawakami et al., 2006; Ross et al., 2003, which are all incorporated byreference herein. For example, elevated levels of inflammatorycytokines, including TNF, interferon-γ, and IL-6, are associated withmajor mental illness (Dickerson et al., 2007). Microglial activation hasalso been linked to major mental illness. Therefore, downregulatinginflammatory cytokines and inhibiting excessive activation of microgliacould be beneficial in patients with schizophrenia, major depression,bipolar disorder, autism-spectrum disorders, and other neuropsychiatricdisorders.

Accordingly, in pathologies involving oxidative stress alone oroxidative stress exacerbated by inflammation, treatment may compriseadministering to a subject a therapeutically effective amount of acompound of this invention, such as those described above or throughoutthis specification. Treatment may be administered preventively, inadvance of a predictable state of oxidative stress (e.g., organtransplantation or the administration of radiation therapy to a cancerpatient), or it may be administered therapeutically in settingsinvolving established oxidative stress and inflammation.

The compounds of the invention may be generally applied to the treatmentof inflammatory conditions, such as sepsis, dermatitis, autoimmunedisease and osteoarthritis. In one aspect, the compounds of thisinvention may be used to treat inflammatory pain and/or neuropathicpain, for example, by inducing Nrf2 and/or inhibiting NF-κB.

In one aspect, the compounds of the invention may be used to function asantioxidant inflammation modulators (AIMs) having potentanti-inflammatory properties that mimic the biological activity ofcyclopentenone prostaglandins (cyPGs). In one embodiment, the compoundsof the invention may be used to control the production ofpro-inflammatory cytokines by selectively targeting regulatory cysteineresidues (RCRs) on proteins that regulate the transcriptional activityof redox-sensitive transcription factors. Activation of RCRs by cyPGs orAIMs has been shown to initiate a pro-resolution program in which theactivity of the antioxidant and cytoprotective transcription factor Nrf2is potently induced, and the activities of the pro-oxidant andpro-inflammatory transcription factors NF-κB and the STATs aresuppressed. This increases the production of antioxidant and reductivemolecules (e.g., NQO1, HO-1, SOD1, and/or γ-GCS) and/or decreasesoxidative stress and the production of pro-oxidant and pro-inflammatorymolecules (e.g., iNOS, COX-2, and/or TNF-α).

In some embodiments, the compounds of the invention may be used in thetreatment and prevention of diseases such as cancer, inflammation,Alzheimer's disease, Parkinson's disease, multiple sclerosis, autism,amyotrophic lateral sclerosis, autoimmune diseases such as rheumatoidarthritis, lupus, and MS, inflammatory bowel disease, all other diseaseswhose pathogenesis is believed to involve excessive production of eithernitric oxide or prostaglandins, and pathologies involving oxidativestress alone or oxidative stress exacerbated by inflammation.

Another aspect of inflammation is the production of inflammatoryprostaglandins such as prostaglandin E. These molecules promotevasodilation, plasma extravasation, localized pain, elevatedtemperature, and other symptoms of inflammation. The inducible form ofthe enzyme COX-2 is associated with their production, and high levels ofCOX-2 are found in inflamed tissues. Consequently, inhibition of COX-2may relieve many symptoms of inflammation and a number of importantanti-inflammatory drugs (e.g., ibuprofen and celecoxib) act byinhibiting COX-2 activity. Recent research, however, has demonstratedthat a class of cyclopentenone prostaglandins (cyPGs) (e.g., 15-deoxyprostaglandin J2, a.k.a. PGJ2) plays a role in stimulating theorchestrated resolution of inflammation (e.g., Rajakariar et al., 2007).COX-2 is also associated with the production of cyclopentenoneprostaglandins. Consequently, inhibition of COX-2 may interfere with thefull resolution of inflammation, potentially promoting the persistenceof activated immune cells in tissues and leading to chronic,“smoldering” inflammation. This effect may be responsible for theincreased incidence of cardiovascular disease in patients usingselective COX-2 inhibitors for long periods of time.

In one aspect, the compounds of the invention may be used to control theproduction of pro-inflammatory cytokines within the cell by selectivelyactivating regulatory cysteine residues (RCRs) on proteins that regulatethe activity of redox-sensitive transcription factors. Activation ofRCRs by cyPGs has been shown to initiate a pro-resolution program inwhich the activity of the antioxidant and cytoprotective transcriptionfactor Nrf2 is potently induced and the activities of the pro-oxidantand pro-inflammatory transcription factors NF-κB and the STATs aresuppressed. In some embodiments, this increases the production ofantioxidant and reductive molecules (NQO1, HO-1, SOD1, γ-GCS) anddecreases oxidative stress and the production of pro-oxidant andpro-inflammatory molecules (iNOS, COX-2, TNF-α). In some embodiments,the compounds of this invention may cause the cells that host theinflammatory event to revert to a non-inflammatory state by promotingthe resolution of inflammation and limiting excessive tissue damage tothe host.

A. Cancer

Further, the compounds of the present disclosure may be used to induceapoptosis in tumor cells, to induce cell differentiation, to inhibitcancer cell proliferation, to inhibit an inflammatory response, and/orto function in a chemopreventative capacity. For example, the inventionprovides new compounds that have one or more of the followingproperties: (1) an ability to induce apoptosis and differentiate bothmalignant and non-malignant cells, (2) an activity at sub-micromolar ornanomolar levels as an inhibitor of proliferation of many malignant orpremalignant cells, (3) an ability to suppress the de novo synthesis ofthe inflammatory enzyme inducible nitric oxide synthase (iNOS), (4) anability to inhibit NF-κB activation, and (5) an ability to induce theexpression of heme oxygenase-1 (HO-1).

The levels of iNOS and COX-2 are elevated in certain cancers and havebeen implicated in carcinogenesis and COX-2 inhibitors have been shownto reduce the incidence of primary colonic adenomas in humans (Rostom etal., 2007; Brown and DuBois, 2005; Crowel et al., 2003). iNOS isexpressed in myeloid-derived suppressor cells (MDSCs) (Angulo et al.,2000) and COX-2 activity in cancer cells has been shown to result in theproduction of prostaglandin E₂ (PGE₂), which has been shown to inducethe expression of arginase in MDSCs (Sinha et al., 2007). Arginase andiNOS are enzymes that utilize L-arginine as a substrate and produceL-ornithine and urea, and L-citrulline and NO, respectively. Thedepletion of arginine from the tumor microenvironment by MDSCs, combinedwith the production of NO and peroxynitrite has been shown to inhibitproliferation and induce apoptosis of T cells (Bronte et al., 2003)Inhibition of COX-2 and iNOS has been shown to reduce the accumulationof MDSCs, restore cytotoxic activity of tumor-associated T cells, anddelay tumor growth (Sinha et al., 2007; Mazzoni et al., 2002; Zhou etal., 2007).

Inhibition of the NF-κB and JAK/STAT signaling pathways has beenimplicated as a strategy to inhibit proliferation of cancer epithelialcells and induce their apoptosis. Activation of STAT3 and NF-κB has beenshown to result in suppression of apoptosis in cancer cells, andpromotion of proliferation, invasion, and metastasis. Many of the targetgenes involved in these processes have been shown to betranscriptionally regulated by both NF-κB and STAT3 (Yu et al., 2007).

In addition to their direct roles in cancer epithelial cells, NF-κB andSTAT3 also have important roles in other cells found within the tumormicroenvironment. Experiments in animal models have demonstrated thatNF-κB is required in both cancer cells and hematopoeitic cells topropagate the effects of inflammation on cancer initiation andprogression (Greten et al., 2004). NF-κB inhibition in cancer andmyeloid cells reduces the number and size, respectively, of theresultant tumors. Activation of STAT3 in cancer cells results in theproduction of several cytokines (IL-6, IL-10) which suppress thematuration of tumor-associated dendritic cells (DC). Furthermore, STAT3is activated by these cytokines in the dendritic cells themselvesInhibition of STAT3 in mouse models of cancer restores DC maturation,promotes antitumor immunity, and inhibits tumor growth (Kortylewski etal., 2005).

B. Treatment of Multiple Sclerosis

The compounds and methods of this invention may be used for treatingpatients for multiple sclerosis (MS). MS is known to be an inflammatorycondition of the central nervous system (Williams et al., 1994; Merrilland Benvenist, 1996; Genain and Nauser, 1997). Based on severalinvestigations, there is evidence suggesting that inflammatory,oxidative, and/or immune mechanisms are involved in the pathogenesis ofAlzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateralsclerosis (ALS), and MS (Bagasra et al., 1995; McGeer and McGeer, 1995;Simonian and Coyle, 1996; Kaltschmidt et al., 1997). Both reactiveastrocytes and activated microglia have been implicated in causation ofneurodegenerative disease (NDD) and neuroinflammatory disease (NID);there has been a particular emphasis on microglia as cells thatsynthesize both NO and prostaglandins as products of the respectiveenzymes, iNOS and COX-2. De novo formation of these enzymes may bedriven by inflammatory cytokines such as interferon-γ or interleukin-1.In turn, excessive production of NO may lead to inflammatory cascadesand/or oxidative damage in cells and tissues of many organs, includingneurons and oligodendrocytes of the nervous system, with consequentmanifestations in AD and MS, and possible PD and ALS (Coyle andPuttfarcken, 1993; Beal, 1996; Merrill and Benvenist, 1996; Simonian andCoyle, 1996; Vodovotz et al., 1996). Epidemiologic data indicate thatchronic use of NSAID's which block synthesis of prostaglandins fromarachidonate, markedly lower the risk for development of AD (McGeer etal., 1996; Stewart et al., 1997). Thus, agents that block formation ofNO and prostaglandins, may be used in approaches to prevention andtreatment of NDD. Successful therapeutic candidates for treating such adisease typically require an ability to penetrate the blood-brainbarrier. See, for example, U.S. Patent Publication 2009/0060873, whichis incorporated by reference herein in its entirety.

C. Neuroinflammation

Neuroinflammation encapsulates the idea that microglial and astrocyticresponses and actions in the central nervous system have a fundamentallyinflammation-like character, and that these responses are central to thepathogenesis and progression of a wide variety of neurologicaldisorders. This idea originated in the field of Alzheimer's disease(Griffin et al., 1989; Rogers et al., 1988), where it has revolutionizedour understanding of this disease (Akiyama et al., 2000). These ideashave been extended to other neurodegenerative diseases (Eikelenboom etal., 2002; Ishizawa and Dickson, 2001), to ischemic/toxic diseases(Gehrmann et al., 1995; Touzani et al., 1999), to tumor biology (Graeberet al., 2002) and even to normal brain development.

Neuroinflammation incorporates a wide spectrum of complex cellularresponses that include activation of microglia and astrocytes andinduction of cytokines, chemokines, complement proteins, acute phaseproteins, oxidative injury, and related molecular processes. Theseevents may have detrimental effects on neuronal function, leading toneuronal injury, further glial activation, and ultimatelyneurodegeneration.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with neuroinflammation.

D. Treatment of Renal Failure

Another aspect of the present disclosure concerns new methods andcompounds for the treatment and prevention of renal disease. See U.S.patent application Ser. No. 12/352,473, which is incorporated byreference herein in its entirety. Renal failure, resulting in inadequateclearance of metabolic waste products from the blood and abnormalconcentrations of electrolytes in the blood, is a significant medicalproblem throughout the world, especially in developed countries.Diabetes and hypertension are among the most important causes of chronicrenal failure (CKD), but it is also associated with other conditionssuch as lupus. Acute renal failure may arise from exposure to certaindrugs (e.g., acetaminophen) or toxic chemicals, or fromischemia-reperfusion injury associated with shock or surgical proceduressuch as transplantation, and may result in chronic renal failure. Inmany patients, renal failure advances to a stage in which the patientrequires regular dialysis or kidney transplantation to continue living.Both of these procedures are highly invasive and associated withsignificant side effects and quality of life issues. Although there areeffective treatments for some complications of renal failure, such ashyperparathyroidism and hyperphosphatemia, no available treatment hasbeen shown to halt or reverse the underlying progression of renalfailure. Thus, agents that can improve compromised renal function wouldrepresent a significant advance in the treatment of renal failure.

Inflammation contributes significantly to the pathology of CKD. There isalso a strong mechanistic link between oxidative stress and renaldysfunction. The NF-κB signaling pathway plays an important role in theprogression of CKD as NF-κB regulates the transcription of MCP-1, achemokine that is responsible for the recruitment ofmonocytes/macrophages resulting in an inflammatory response thatultimately injures the kidney (Wardle, 2001). The Keap1/Nrf2/ARE pathwaycontrols the transcription of several genes encoding antioxidantenzymes, including heme oxygenase-1 (HO-1). Ablation of the Nrf2 gene infemale mice results in the development of lupus-like glomerularnephritis (Yoh et al., 2001). Furthermore, several studies havedemonstrated that HO-1 expression is induced in response to renal damageand inflammation and that this enzyme and its products—bilirubin andcarbon monoxide—play a protective role in the kidney (Nath et al.,2006).

The glomerulus and the surrounding Bowman's capsule constitute the basicfunctional unit of the kidney. Glomerular filtration rate (GFR) is thestandard measure of renal function. Creatinine clearance is commonlyused to measure GFR. However, the level of serum creatinine is commonlyused as a surrogate measure of creatinine clearance. For instance,excessive levels of serum creatinine are generally accepted to indicateinadequate renal function and reductions in serum creatinine over timeare accepted as an indication of improved renal function. Normal levelsof creatinine in the blood are approximately 0.6 to 1.2 milligrams (mg)per deciliter (dl) in adult males and 0.5 to 1.1 milligrams perdeciliter in adult females.

Acute kidney injury (AKI) can occur following ischemia-reperfusion,treatment with certain pharmacological agents such as cisplatin andrapamycin, and intravenous injection of radiocontrast media used inmedical imaging. As in CKD, inflammation and oxidative stress contributeto the pathology of AKI. The molecular mechanisms underlyingradiocontrast-induced nephropathy (RCN) are not well understood;however, it is likely that a combination of events including prolongedvasoconstriction, impaired kidney autoregulation, and direct toxicity ofthe contrast media all contribute to renal failure (Tumlin et al.,2006). Vasoconstriction results in decreased renal blood flow and causesischemia-reperfusion and the production of reactive oxygen species. HO-1is strongly induced under these conditions and has been demonstrated toprevent ischemia-reperfusion injury in several different organs,including the kidney (Nath et al., 2006). Specifically, induction ofHO-1 has been shown to be protective in a rat model of RCN (Goodman etal., 2007). Reperfusion also induces an inflammatory response, in partthough activation of NF-κB signaling (Nichols, 2004). Targeting NF-κBhas been proposed as a therapeutic strategy to prevent organ damage(Zingarelli et al., 2003).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with renal failure.

E. Cardiovascular Disease

The compounds and methods of this invention may be used for treatingpatients with cardiovascular disease. See U.S. patent application Ser.No. 12/352,473, which is incorporated by reference herein in itsentirety. Cardiovascular (CV) disease is among the most important causesof mortality worldwide, and is the leading cause of death in manydeveloped nations. The etiology of CV disease is complex, but themajority of causes are related to inadequate or completely disruptedsupply of blood to a critical organ or tissue. Frequently such acondition arises from the rupture of one or more atheroscleroticplaques, which leads to the formation of a thrombus that blocks bloodflow in a critical vessel. Such thrombosis is the principal cause ofheart attacks, in which one or more of the coronary arteries is blockedand blood flow to the heart itself is disrupted. The resulting ischemiais highly damaging to cardiac tissue, both from lack of oxygen duringthe ischemic event and from excessive formation of free radicals afterblood flow is restored (a phenomenon known as ischemia-reperfusioninjury). Similar damage occurs in the brain during a thrombotic stroke,when a cerebral artery or other major vessel is blocked by thrombosis.Hemorrhagic strokes, in contrast, involve rupture of a blood vessel andbleeding into the surrounding brain tissue. This creates oxidativestress in the immediate area of the hemorrhage, due to the presence oflarge amounts of free heme and other reactive species, and ischemia inother parts of the brain due to compromised blood flow. Subarachnoidhemorrhage, which is frequently accompanied by cerebral vasospasm, alsocauses ischemia/reperfusion injury in the brain.

Alternatively, atherosclerosis may be so extensive in critical bloodvessels that stenosis (narrowing of the arteries) develops and bloodflow to critical organs (including the heart) is chronicallyinsufficient. Such chronic ischemia can lead to end-organ damage of manykinds, including the cardiac hypertrophy associated with congestiveheart failure.

Atherosclerosis, the underlying defect leading to many forms ofcardiovascular disease, occurs when a physical defect or injury to thelining (endothelium) of an artery triggers an inflammatory responseinvolving the proliferation of vascular smooth muscle cells and theinfiltration of leukocytes into the affected area. Ultimately, acomplicated lesion known as an atherosclerotic plaque may form, composedof the above-mentioned cells combined with deposits ofcholesterol-bearing lipoproteins and other materials (e.g., Hansson etal., 2006).

Pharmaceutical treatments for cardiovascular disease include preventivetreatments, such as the use of drugs intended to lower blood pressure orcirculating levels of cholesterol and lipoproteins, as well astreatments designed to reduce the adherent tendencies of platelets andother blood cells (thereby reducing the rate of plaque progression andthe risk of thrombus formation). More recently, drugs such asstreptokinase and tissue plasminogen activator have been introduced andare used to dissolve the thrombus and restore blood flow. Surgicaltreatments include coronary artery bypass grafting to create analternative blood supply, balloon angioplasty to compress plaque tissueand increase the diameter of the arterial lumen, and carotidendarterectomy to remove plaque tissue in the carotid artery. Suchtreatments, especially balloon angioplasty, may be accompanied by theuse of stents, expandable mesh tubes designed to support the arterywalls in the affected area and keep the vessel open. Recently, the useof drug-eluting stents has become common in order to preventpost-surgical restenosis (renarrowing of the artery) in the affectedarea. These devices are wire stents coated with a biocompatible polymermatrix containing a drug that inhibits cell proliferation (e.g.,paclitaxel or rapamycin). The polymer allows a slow, localized releaseof the drug in the affected area with minimal exposure of non-targettissues. Despite the significant benefits offered by such treatments,mortality from cardiovascular disease remains high and significant unmetneeds in the treatment of cardiovascular disease remain.

As noted above, induction of HO-1 has been shown to be beneficial in avariety of models of cardiovascular disease, and low levels of HO-1expression have been clinically correlated with elevated risk of CVdisease. Compounds of the invention, therefore, may be used in treatingor preventing a variety of cardiovascular disorders including but notlimited to atherosclerosis, hypertension, myocardial infarction, chronicheart failure, stroke, subarachnoid hemorrhage, and restenosis.

F. Diabetes

Diabetes is a complex disease characterized by the body's failure toregulate circulating levels of glucose. See U.S. patent application Ser.No. 12/352,473, which is incorporated by reference herein in itsentirety. This failure may result from a lack of insulin, a peptidehormone that regulates the both the production and absorption of glucosein various tissues. Deficient insulin compromises the ability of muscle,fat, and other tissues to absorb glucose properly, leading tohyperglycemia (abnormally high levels of glucose in the blood). Mostcommonly, such insulin deficiency results from inadequate production inthe islet cells of the pancreas. In the majority of cases this arisesfrom autoimmune destruction of these cells, a condition known as type 1or juvenile-onset diabetes, but may also be due to physical trauma orsome other cause.

Diabetes may also arise when muscle and fat cells become less responsiveto insulin and do not absorb glucose properly, resulting inhyperglycemia. This phenomenon is known as insulin resistance, and theresulting condition is known as Type 2 diabetes. Type 2 diabetes, themost common type, is highly associated with obesity and hypertension.Obesity is associated with an inflammatory state of adipose tissue thatis thought to play a major role in the development of insulin resistance(e.g., Hotamisligil, 2006; Guilherme et al., 2008).

Diabetes is associated with damage to many tissues, largely becausehyperglycemia (and hypoglycemia, which can result from excessive orpoorly timed doses of insulin) is a significant source of oxidativestress. Chronic kidney failure, retinopathy, peripheral neuropathy,peripheral vasculitis, and the development of dermal ulcers that healslowly or not at all are among the common complications of diabetes.Because of their ability to protect against oxidative stress,particularly by the induction of HO-1 expression, compounds of theinvention may be used in treatments for many complications of diabetes.As noted above (Cai et al., 2005), chronic inflammation and oxidativestress in the liver are suspected to be primary contributing factors inthe development of Type 2 diabetes. Furthermore, PPARγ agonists such asthiazolidinediones are capable of reducing insulin resistance and areknown to be effective treatments for Type 2 diabetes.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with neuroinflammation.

The effect of treatment of diabetes may be evaluated as follows. Boththe biological efficacy of the treatment modality as well as theclinical efficacy are evaluated, if possible. For example, diseasemanifests itself by increased blood sugar, the biological efficacy ofthe treatment therefore can be evaluated, for example, by observation ofreturn of the evaluated blood glucose towards normal. Measuring aclinical endpoint which can give an indication of b-cell regenerationafter, for example, a six-month period of time, can give an indicationof the clinical efficacy of the treatment regimen.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with diabetes.

G. Rheumatoid Arthritis

The compounds and methods of this invention may be used for treatingpatients with RA. Typically the first signs of rheumatoid arthritis (RA)appear in the synovial lining layer, with proliferation of synovialfibroblasts and their attachment to the articular surface at the jointmargin (Lipsky, 1998). Subsequently, macrophages, T cells and otherinflammatory cells are recruited into the joint, where they produce anumber of mediators, including the cytokines interleukin-1 (IL-1), whichcontributes to the chronic sequelae leading to bone and cartilagedestruction, and tumour necrosis factor (TNF-α), which plays a role ininflammation (Dinarello, 1998; Arend and Dayer, 1995; van den Berg,2001). The concentration of IL-1 in plasma is significantly higher inpatients with RA than in healthy individuals and, notably, plasma IL-1levels correlate with RA disease activity (Eastgate et al., 1988).Moreover, synovial fluid levels of IL-1 are correlated with variousradiographic and histologic features of RA (Kahle et al., 1992; Rooneyet al., 1990).

In normal joints, the effects of these and other proinflammatorycytokines are balanced by a variety of anti-inflammatory cytokines andregulatory factors (Burger and Dayer, 1995). The significance of thiscytokine balance is illustrated in juvenile RA patients, who havecyclical increases in fever throughout the day (Prieur et al., 1987).After each peak in fever, a factor that blocks the effects of IL-1 isfound in serum and urine. This factor has been isolated, cloned andidentified as IL-1 receptor antagonist (IL-1ra), a member of the IL-1gene family (Hannum et al., 1990). IL-1ra, as its name indicates, is anatural receptor antagonist that competes with IL-1 for binding to typeI IL-1 receptors and, as a result, blocks the effects of IL-1 (Arend etal., 1998). A 10- to 100-fold excess of IL-1ra may be needed to blockIL-1 effectively; however, synovial cells isolated from patients with RAdo not appear to produce enough IL-1ra to counteract the effects of IL-1(Firestein et al., 1994; Fujikawa et al., 1995).

H. Psoriatic Arthritis

Psoriasis is an inflammatory and proliferative skin disorder with aprevalence of 1.5-3%. Approximately 20% of patients with psoriasisdevelop a characteristic form of arthritis that has several patterns(Gladman, 1992; Jones et al., 1994; Gladman et al., 1995). Someindividuals present with joint symptoms first but in the majority, skinpsoriasis presents first. About one-third of patients have simultaneousexacerbations of their skin and joint disease (Gladman et al., 1987) andthere is a topographic relationship between nail and distalinterphalangeal joint disease (Jones et al., 1994; Wright, 1956).Although the inflammatory processes which link skin, nail and jointdisease remain elusive, an immune-mediated pathology is implicated.

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathycharacterized by the association of arthritis and psoriasis and wasrecognized as a clinical entity distinct from rheumatoid arthritis (RA)in 1964 (Blumberg et al., 1964). Subsequent studies have revealed thatPsA shares a number of genetic, pathogenic and clinical features withother spondyloarthropathies (SpAs), a group of diseases that compriseankylosing spondylitis, reactive arthritis and enteropathic arthritis(Wright, 1979). The notion that PsA belongs to the SpA group hasrecently gained further support from imaging studies demonstratingwidespread enthesitis in the, including PsA but not RA (McGonagle etal., 1999; McGonagle et al., 1998). More specifically, enthesitis hasbeen postulated to be one of the earliest events occurring in the SpAs,leading to bone remodeling and ankylosis in the spine, as well as toarticular synovitis when the inflamed entheses are close to peripheraljoints. However, the link between enthesitis and the clinicalmanifestations in PsA remains largely unclear, as PsA can present withfairly heterogeneous patterns of joint involvement with variable degreesof severity (Marsal et al., 1999; Salvarani et al., 1998). Thus, otherfactors must be posited to account for the multifarious features of PsA,only a few of which (such as the expression of the HLA-B27 molecule,which is strongly associated with axial disease) have been identified.As a consequence, it remains difficult to map the disease manifestationsto specific pathogenic mechanisms, which means that the treatment ofthis condition remains largely empirical.

Family studies have suggested a genetic contribution to the developmentof PsA (Moll and Wright, 1973). Other chronic inflammatory forms ofarthritis, such as ankylosing spondylitis and rheumatoid arthritis, arethought to have a complex genetic basis. However, the genetic componentof PsA has been difficult to assess for several reasons. There is strongevidence for a genetic predisposition to psoriasis alone that may maskthe genetic factors that are important for the development of PsA.Although most would accept PsA as a distinct disease entity, at timesthere is a phenotypic overlap with rheumatoid arthritis and ankylosingspondylitis. Also, PsA itself is not a homogeneous condition and varioussubgroups have been proposed.

Increased amounts of TNF-α have been reported in both psoriatic skin(Ettehadi et al., 1994) and synovial fluid (Partsch et al., 1997).Recent trials have shown a positive benefit of anti-TNF treatment inboth PsA (Mease et al., 2000) and ankylosing spondylitis (Brandt et al.,2000).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with psoriatic arthritis.

I. Reactive Arthritis

In reactive arthritis (ReA) the mechanism of joint damage is unclear,but it is likely that cytokines play critical roles. A more prevalentTh1 profile high levels of interferon gamma (IFN-γ) and low levels ofinterleukin 4 (IL-4) has been reported (Lahesmaa et al., 1992; Schlaaket al., 1992; Simon et al., 1993; Schlaak et al., 1996; Kotake et al.,1999; Ribbens et al., 2000), but several studies have shown relativepredominance of IL-4 and IL-10 and relative lack of IFN-γ and tumournecrosis factor alpha (TNF-α) in the synovial membrane (Simon et al.,1994; Yin et al., 1999) and fluid (SF) (Yin et al., 1999; Yin et al.,1997) of reactive arthritis patients compared with rheumatoid arthritis(RA) patients. A lower level of TNF-α secretion in reactive arthritisthan in RA patients has also been reported after ex vivo stimulation ofperipheral blood mononuclear cells (PBMC) (Braun et al., 1999).

It has been argued that clearance of reactive arthritis-associatedbacteria requires the production of appropriate levels of IFN-γ andTNF-α, while IL-10 acts by suppressing these responses (Autenrieth etal., 1994; Sieper and Braun, 1995). IL-10 is a regulatory cytokine thatinhibits the synthesis of IL-12 and TNF-γ by activated macrophages (deWaal et al., 1991; Hart et al., 1995; Chomarat et al., 1995) and ofIFN-γ by T cells (Macatonia et al., 1993).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with reactive arthritis.

J. Enteropathic Arthritis

Typically enteropathic arthritis (EA) occurs in combination withinflammatory bowel diseases (IBD) such as Crohn's disease or ulcerativecolitis. It also can affect the spine and sacroiliac joints.Enteropathic arthritis involves the peripheral joints, usually in thelower extremities such as the knees or ankles. It commonly involves onlya few or a limited number of joints and may closely follow the bowelcondition. This occurs in approximately 11% of patients with ulcerativecolitis and 21% of those with Crohn's disease. The synovitis isgenerally self-limited and non-deforming.

Enteropathic arthropathies comprise a collection of rheumatologicconditions that share a link to GI pathology. These conditions includereactive (i.e., infection-related) arthritis due to bacteria (e.g.,Shigella, Salmonella, Campylobacter, Yersinia species, Clostridiumdifficile), parasites (e.g., Strongyloides stercoralis, Taenia saginata,Giardia lamblia, Ascaris lumbricoides, Cryptosporidium species), andspondyloarthropathies associated with inflammatory bowel disease (IBD).Other conditions and disorders include intestinal bypass (jejunoileal),arthritis, celiac disease, Whipple disease, and collagenous colitis.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with enteropathic arthritis.

K. Juvenile Rheumatoid Arthritis

Juvenile rheumatoid arthritis (JRA), a term for the most prevalent formof arthritis in children, is applied to a family of illnessescharacterized by chronic inflammation and hypertrophy of the synovialmembranes. The term overlaps, but is not completely synonymous, with thefamily of illnesses referred to as juvenile chronic arthritis and/orjuvenile idiopathic arthritis in Europe.

Both innate and adaptive immune systems use multiple cell types, a vastarray of cell surface and secreted proteins, and interconnected networksof positive and negative feedback (Lo et al., 1999). Furthermore, whileseparable in thought, the innate and adaptive wings of the immune systemare functionally intersected (Fearon and Locksley, 1996), and pathologicevents occurring at these intersecting points are likely to be highlyrelevant to our understanding of pathogenesis of adult and childhoodforms of chronic arthritis (Warrington, et al., 2001).

Polyarticular JRA is a distinct clinical subtype characterized byinflammation and synovial proliferation in multiple joints (four ormore), including the small joints of the hands (Jarvis, 2002). Thissubtype of JRA may be severe, because of both its multiple jointinvolvement and its capacity to progress rapidly over time. Althoughclinically distinct, polyarticular JRA is not homogeneous, and patientsvary in disease manifestations, age of onset, prognosis, and therapeuticresponse. These differences very likely reflect a spectrum of variationin the nature of the immune and inflammatory attack that can occur inthis disease (Jarvis, 1998).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with JRA.

L. Early Inflammatory Arthritis

The compounds and methods of this invention may be used for treatingpatients with early inflammatory arthritis. The clinical presentation ofdifferent inflammatory arthropathies is similar early in the course ofdisease. As a result, it is often difficult to distinguish patients whoare at risk of developing the severe and persistent synovitis that leadsto erosive joint damage from those whose arthritis is more self-limited.Such distinction is critical in order to target therapy appropriately,treating aggressively those with erosive disease and avoidingunnecessary toxicity in patients with more self-limited disease. Currentclinical criteria for diagnosing erosive arthropathies such asrheumatoid arthritis (RA) are less effective in early disease andtraditional markers of disease activity such as joint counts and acutephase response do not adequately identify patients likely to have pooroutcomes (Harrison et al., 1998). Parameters reflective of thepathologic events occurring in the synovium are most likely to be ofsignificant prognostic value.

Recent efforts to identify predictors of poor outcome in earlyinflammatory arthritis have identified the presence of RA specificautoantibodies, in particular antibodies towards citrullinated peptides,to be associated with erosive and persistent disease in earlyinflammatory arthritis cohorts. On the basis of this, a cyclicalcitrullinated peptide (CCP) has been developed to assist in theidentification of anti-CCP antibodies in patient sera. Using thisapproach, the presence of anti-CCP antibodies has been shown to bespecific and sensitive for RA, can distinguish RA from otherarthropathies, and can potentially predict persistent, erosive synovitisbefore these outcomes become clinically manifest. Importantly, anti-CCPantibodies are often detectable in sera many years prior to clinicalsymptoms suggesting that they may be reflective of subclinical immuneevents (Nielen et al., 2004; Rantapaa-Dahlqvist et al., 2003).

M. Ankylosing Spondylitis

AS is a disease subset within a broader disease classification ofspondyloarthropathy. Patients affected with the various subsets ofspondyloarthropathy have disease etiologies that are often verydifferent, ranging from bacterial infections to inheritance. Yet, in allsubgroups, the end result of the disease process is axial arthritis.Despite the early clinically differences seen in the various patientpopulations, many of them end up nearly identical after a disease courseof ten-to-twenty years. Recent studies suggest the mean time to clinicaldiagnosis of ankylosing spondylitis from disease onset of disease is 7.5years (Khan, 1998). These same studies suggest that thespondyloarthropathies may have prevalence close to that of rheumatoidarthritis (Feldtkeller et al., 2003; Doran et al., 2003).

AS is a chronic systemic inflammatory rheumatic disorder of the axialskeleton with or without extraskeletal manifestations. Sacroiliac jointsand the spine are primarily affected, but hip and shoulder joints, andless commonly peripheral joints or certain extra-articular structuressuch as the eye, vasculature, nervous system, and gastrointestinalsystem may also be involved. Its etiology is not yet fully understood(Wordsworth, 1995; Calin and Taurog, 1998). It is strongly associatedwith the major histocompatibility class I (MHC I) HLA-B27 allele (Calinand Taurog, 1998). AS affects individuals in the prime of their life andis feared because of its potential to cause chronic pain andirreversible damage of tendons, ligaments, joints, and bones (Brewertonet al., 1973a; Brewerton et al., 1973b; Schlosstein et al., 1973). ASmay occur alone or in association with another form ofspondyloarthropathy such as reactive arthritis, psoriasis, psoriaticarthritis, enthesitis, ulcerative colitis, irritable bowel disease, orCrohn's disease, in which case it is classified as secondary AS.

Typically, the affected sites include the discovertebral, apophyseal,costovertebral, and costotransverse joints of the spine, and theparavertebral ligamentous structures. Inflammation of the entheses,which are sites of musculotendinous and ligamentous attachment to bones,is also prominent in this disease (Calin and Taurog, 1998). The site ofenthesitis is known to be infiltrated by plasma cells, lymphocytes, andpolymorphonuclear cells. The inflammatory process frequently results ingradual fibrous and bony ankylosis, (Ball, 1971; Khan, 1990).

Delayed diagnosis is common because symptoms are often attributed tomore common back problems. A dramatic loss of flexibility in the lumbarspine is an early sign of AS. Other common symptoms include chronic painand stiffness in the lower back which usually starts where the lowerspine is joined to the pelvis, or hip. Although most symptoms begin inthe lumbar and sacroiliac areas, they may involve the neck and upperback as well. Arthritis may also occur in the shoulder, hips and feet.Some patients have eye inflammation, and more severe cases must beobserved for heart valve involvement.

The most frequent presentation is back pain, but disease can beginatypically in peripheral joints, especially in children and women, andrarely with acute iritis (anterior uveitis). Additional early symptomsand signs are diminished chest expansion from diffuse costovertebralinvolvement, low-grade fever, fatigue, anorexia, weight loss, andanemia. Recurrent back pain—often nocturnal and of varying intensity—isan eventual complaint, as is morning stiffness typically relieved byactivity. A flexed or bent-over posture eases back pain and paraspinalmuscle spasm; thus, some degree of kyphosis is common in untreatedpatients.

Systemic manifestations occur in ⅓ of patients. Recurrent, usuallyself-limited, acute iritis (anterior uveitis) rarely is protracted andsevere enough to impair vision. Neurologic signs can occasionally resultfrom compression radiculitis or sciatica, vertebral fracture orsubluxation, and cauda equina syndrome (which consists of impotence,nocturnal urinary incontinence, diminished bladder and rectal sensation,and absence of ankle jerks). Cardiovascular manifestations can includeaortic insufficiency, angina, pericarditis, and ECG conductionabnormalities. A rare pulmonary finding is upper lobe fibrosis,occasionally with cavitation that may be mistaken for TB and can becomplicated by infection with Aspergillus.

AS is characterized by mild or moderate flares of active spondylitisalternating with periods of almost or totally inactive inflammation.Proper treatment in most patients results in minimal or no disabilityand in full, productive lives despite back stiffness. Occasionally, thecourse is severe and progressive, resulting in pronounced incapacitatingdeformities. The prognosis is bleak for patients with refractory iritisand for the rare patient with secondary amyloidosis.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with ankylosing spondylitis.

N. Ulcerative Colitis

Ulcerative colitis is a disease that causes inflammation and sores,called ulcers, in the lining of the large intestine. The inflammationusually occurs in the rectum and lower part of the colon, but it mayaffect the entire colon. Ulcerative colitis rarely affects the smallintestine except for the end section, called the terminal ileum.Ulcerative colitis may also be called colitis or proctitis. Theinflammation makes the colon empty frequently, causing diarrhea. Ulcersform in places where the inflammation has killed the cells lining thecolon; the ulcers bleed and produce pus.

Ulcerative colitis is an inflammatory bowel disease (IBD), the generalname for diseases that cause inflammation in the small intestine andcolon. Ulcerative colitis can be difficult to diagnose because itssymptoms are similar to other intestinal disorders and to another typeof IBD, Crohn's disease. Crohn's disease differs from ulcerative colitisbecause it causes inflammation deeper within the intestinal wall. Also,Crohn's disease usually occurs in the small intestine, although it canalso occur in the mouth, esophagus, stomach, duodenum, large intestine,appendix, and anus.

Ulcerative colitis may occur in people of any age, but most often itstarts between ages 15 and 30, or less frequently between ages 50 and70. Children and adolescents sometimes develop the disease. Ulcerativecolitis affects men and women equally and appears to run in somefamilies. Theories about what causes ulcerative colitis abound, but nonehave been proven. The most popular theory is that the body's immunesystem reacts to a virus or a bacterium by causing ongoing inflammationin the intestinal wall. People with ulcerative colitis haveabnormalities of the immune system, but doctors do not know whetherthese abnormalities are a cause or a result of the disease. Ulcerativecolitis is not caused by emotional distress or sensitivity to certainfoods or food products, but these factors may trigger symptoms in somepeople.

The most common symptoms of ulcerative colitis are abdominal pain andbloody diarrhea. Patients also may experience fatigue, weight loss, lossof appetite, rectal bleeding, and loss of body fluids and nutrients.About half of patients have mild symptoms. Others suffer frequent fever,bloody diarrhea, nausea, and severe abdominal cramps. Ulcerative colitismay also cause problems such as arthritis, inflammation of the eye,liver disease (hepatitis, cirrhosis, and primary sclerosingcholangitis), osteoporosis, skin rashes, and anemia. No one knows forsure why problems occur outside the colon. Scientists think thesecomplications may occur when the immune system triggers inflammation inother parts of the body. Some of these problems go away when the colitisis treated.

A thorough physical exam and a series of tests may be required todiagnose ulcerative colitis. Blood tests may be done to check foranemia, which could indicate bleeding in the colon or rectum. Bloodtests may also uncover a high white blood cell count, which is a sign ofinflammation somewhere in the body. By testing a stool sample, thedoctor can detect bleeding or infection in the colon or rectum. Thedoctor may do a colonoscopy or sigmoidoscopy. For either test, thedoctor inserts an endoscope—a long, flexible, lighted tube connected toa computer and TV monitor—into the anus to see the inside of the colonand rectum. The doctor will be able to see any inflammation, bleeding,or ulcers on the colon wall. During the exam, the doctor may do abiopsy, which involves taking a sample of tissue from the lining of thecolon to view with a microscope. A barium enema x ray of the colon mayalso be required. This procedure involves filling the colon with barium,a chalky white solution. The barium shows up white on x-ray film,allowing the doctor a clear view of the colon, including any ulcers orother abnormalities that might be there.

Treatment for ulcerative colitis depends on the seriousness of thedisease. Most people are treated with medication. In severe cases, apatient may need surgery to remove the diseased colon. Surgery is theonly cure for ulcerative colitis. Some people whose symptoms aretriggered by certain foods are able to control the symptoms by avoidingfoods that upset their intestines, like highly seasoned foods, rawfruits and vegetables, or milk sugar (lactose). Each person mayexperience ulcerative colitis differently, so treatment is adjusted foreach individual. Emotional and psychological support is important. Somepeople have remissions—periods when the symptoms go away—that last formonths or even years. However, most patients' symptoms eventuallyreturn. This changing pattern of the disease means one cannot alwaystell when a treatment has helped. Some people with ulcerative colitismay need medical care for some time, with regular doctor visits tomonitor the condition.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with ulcerative colitis.

O. Crohn's Disease

Another disorder for which immunosuppression has been tried is Crohn'sdisease. Crohn's disease symptoms include intestinal inflammation andthe development of intestinal stenosis and fistulas; neuropathy oftenaccompanies these symptoms. Anti-inflammatory drugs, such as5-aminosalicylates (e.g., mesalamine) or corticosteroids, are typicallyprescribed, but are not always effective (reviewed in Botoman et al.,1998). Immunosuppression with cyclosporine is sometimes beneficial forpatients resistant to or intolerant of corticosteroids (Brynskov et al.,1989).

Efforts to develop diagnostic and treatment tools against Crohn'sdisease have focused on the central role of cytokines (Schreiber, 1998;van Hogezand and Verspaget, 1998). Cytokines are small secreted proteinsor factors (5 to 20 kD) that have specific effects on cell-to-cellinteractions, intercellular communication, or the behavior of othercells. Cytokines are produced by lymphocytes, especially T_(H)1 andT_(H)2 lymphocytes, monocytes, intestinal macrophages, granulocytes,epithelial cells, and fibroblasts (reviewed in Rogler and. Andus, 1998;Galley and Webster, 1996). Some cytokines are pro-inflammatory (e.g.,TNF-α, IL-1(α and β), IL-6, IL-8, IL-12, or leukemia inhibitory factor[LIF]); others are anti-inflammatory (e.g., IL-1 receptor antagonist,IL-4, IL-10, IL-11, and TGF-β). However, there may be overlap andfunctional redundancy in their effects under certain inflammatoryconditions.

In active cases of Crohn's disease, elevated concentrations of TNF-α andIL-6 are secreted into the blood circulation, and TNF-α, IL-1, IL-6, andIL-8 are produced in excess locally by mucosal cells (id.; Funakoshi etal., 1998). These cytokines can have far-ranging effects onphysiological systems including bone development, hematopoiesis, andliver, thyroid, and neuropsychiatric function. Also, an imbalance of theIL-1β/IL-1ra ratio, in favor of pro-inflammatory IL-1β, has beenobserved in patients with Crohn's disease (Rogler and Andus, 1998; Saikiet al., 1998; Dionne et al., 1998; but see Kuboyama, 1998). One studysuggested that cytokine profiles in stool samples could be a usefuldiagnostic tool for Crohn's disease (Saiki et al., 1998).

Treatments that have been proposed for Crohn's disease include the useof various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., ofIL-1β converting enzyme and antioxidants) and anti-cytokine antibodies(Rogler and Andus, 1998; van Hogezand and Verspaget, 1998; Reimund etal., 1998; Lugering et al., 1998; McAlindon et al., 1998). Inparticular, monoclonal antibodies against TNF-α have been tried withsome success in the treatment of Crohn's disease (Targan et al., 1997;Stack et al., 1997; van Dullemen et al., 1995). These compounds may beused in combination therapy with compounds of the present disclosure.

Another approach to the treatment of Crohn's disease has focused on atleast partially eradicating the bacterial community that may betriggering the inflammatory response and replacing it with anon-pathogenic community. For example, U.S. Pat. No. 5,599,795 disclosesa method for the prevention and treatment of Crohn's disease in humanpatients. Their method was directed to sterilizing the intestinal tractwith at least one antibiotic and at least one anti-fungal agent to killoff the existing flora and replacing them with different, select,well-characterized bacteria taken from normal humans. Borody taught amethod of treating Crohn's disease by at least partial removal of theexisting intestinal microflora by lavage and replacement with a newbacterial community introduced by fecal inoculum from a disease-screenedhuman donor or by a composition comprising Bacteroides and Escherichiacoli species. (U.S. Pat. No. 5,443,826).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with Crohn's disease.

P. Systemic Lupus Erythematosus

There has also been no known cause for autoimmune diseases such assystemic lupus erythematosus. Systemic lupus erythematosus (SLE) is anautoimmune rheumatic disease characterized by deposition in tissues ofautoantibodies and immune complexes leading to tissue injury (Kotzin,1996). In contrast to autoimmune diseases such as MS and type 1 diabetesmellitus, SLE potentially involves multiple organ systems directly, andits clinical manifestations are diverse and variable (reviewed by Kotzinand O'Dell, 1995). For example, some patients may demonstrate primarilyskin rash and joint pain, show spontaneous remissions, and requirelittle medication. At the other end of the spectrum are patients whodemonstrate severe and progressive kidney involvement that requirestherapy with high doses of steroids and cytotoxic drugs such ascyclophosphamide (Kotzin, 1996).

The serological hallmark of SLE, and the primary diagnostic testavailable, is elevated serum levels of IgG antibodies to constituents ofthe cell nucleus, such as double-stranded DNA (dsDNA), single-strandedDNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNAantibodies play a major role in the development of lupusglomerulonephritis (G N) (Hahn and Tsao, 1993; Ohnishi et al., 1994).Glomerulonephritis is a serious condition in which the capillary wallsof the kidney's blood purifying glomeruli become thickened by accretionson the epithelial side of glomerular basement membranes. The disease isoften chronic and progressive and may lead to eventual renal failure.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with SLE.

Q. Irritable Bowel Syndrome

The compounds and methods of this invention may be used for treatingpatients with Irritable bowel syndrome (IBS). IBS is a functionaldisorder characterized by abdominal pain and altered bowel habits. Thissyndrome may begin in young adulthood and can be associated withsignificant disability. This syndrome is not a homogeneous disorder.Rather, subtypes of IBS have been described on the basis of thepredominant symptom—diarrhea, constipation, or pain. In the absence of“alarm” symptoms, such as fever, weight loss, and gastrointestinalbleeding, a limited workup is needed. Once a diagnosis of IBS is made,an integrated treatment approach can effectively reduce the severity ofsymptoms. IBS is a common disorder, although its prevalence rates havevaried. In general, IBS affects about 15% of US adults and occurs aboutthree times more often in women than in men (Jailwala et al., 2000).

IBS accounts for between 2.4 million and 3.5 million visits tophysicians each year. It not only is the most common condition seen bygastroenterologists but also is one of the most common gastrointestinalconditions seen by primary care physicians (Everhart et al., 1991;Sandler, 1990).

IBS is also a costly disorder. Compared with persons who do not havebowel symptoms, persons with IBS miss three times as many workdays andare more likely to report being too sick to work (Drossman et al., 1993;Drossman et al., 1997). Moreover, those with IBS incur hundreds ofdollars more in medical charges than persons without bowel disorders(Talley et al., 1995).

No specific abnormality accounts for the exacerbations and remissions ofabdominal pain and altered bowel habits experienced by patients withIBS. The evolving theory of IBS suggests dysregulation at multiplelevels of the brain-gut axis. Dysmotility, visceral hypersensitivity,abnormal modulation of the central nervous system (CNS), and infectionhave all been implicated. In addition, psychosocial factors play animportant modifying role. Abnormal intestinal motility has long beenconsidered a factor in the pathogenesis of IBS. Transit time through thesmall intestine after a meal has been shown to be shorter in patientswith diarrhea-predominant IBS than in patients who have theconstipation-predominant or pain-predominant subtype (Cann et al.,1983).

In studies of the small intestine during fasting, the presence of bothdiscrete, clustered contractions and prolonged, propagated contractionshas been reported in patients with IBS (Kellow and Phillips, 1987). Theyalso experience pain with irregular contractions more often than healthypersons (Kellow and Phillips, 1987; Horwitz and Fisher, 2001)

These motility findings do not account for the entire symptom complex inpatients with IBS; in fact, most of these patients do not havedemonstrable abnormalities (Rothstein, 2000). Patients with IBS haveincreased sensitivity to visceral pain. Studies involving balloondistention of the rectosigmoid colon have shown that patients with IBSexperience pain and bloating at pressures and volumes much lower thancontrol subjects (Whitehead et al., 1990). These patients maintainnormal perception of somatic stimuli.

Multiple theories have been proposed to explain this phenomenon. Forexample, receptors in the viscera may have increased sensitivity inresponse to distention or intraluminal contents. Neurons in the dorsalhorn of the spinal cord may have increased excitability. In addition,alteration in CNS processing of sensations may be involved (Drossman etal., 1997). Functional magnetic resonance imaging studies have recentlyshown that compared with control subjects, patients with IBS haveincreased activation of the anterior cingulate cortex, an important paincenter, in response to a painful rectal stimulus (Mertz et al., 2000).

Increasingly, evidence suggests a relationship between infectiousenteritis and subsequent development of IBS. Inflammatory cytokines mayplay a role. In a survey of patients with a history of confirmedbacterial gastroenteritis (Neal et al., 1997), 25% reported persistentalteration of bowel habits. Persistence of symptoms may be due topsychological stress at the time of acute infection (Gwee et al., 1999).

Recent data suggest that bacterial overgrowth in the small intestine mayhave a role in IBS symptoms. In one study (Pimentel et al., 2000), 157(78%) of 202 IBS patients referred for hydrogen breath testing had testfindings that were positive for bacterial overgrowth. Of the 47 subjectswho had follow-up testing, 25 (53%) reported improvement in symptoms(i.e., abdominal pain and diarrhea) with antibiotic treatment.

IBS may present with a range of symptoms. However, abdominal pain andaltered bowel habits remain the primary features. Abdominal discomfortis often described as crampy in nature and located in the left lowerquadrant, although the severity and location can differ greatly.Patients may report diarrhea, constipation, or alternating episodes ofdiarrhea and constipation. Diarrheal symptoms are typically described assmall-volume, loose stools, and stool is sometimes accompanied by mucusdischarge. Patients also may report bloating, fecal urgency, incompleteevacuation, and abdominal distention. Upper gastrointestinal symptoms,such as gastroesophageal reflux, dyspepsia, or nausea, may also bepresent (Lynn and Friedman, 1993).

Persistence of symptoms is not an indication for further testing; it isa characteristic of IBS and is itself an expected symptom of thesyndrome. More extensive diagnostic evaluation is indicated in patientswhose symptoms are worsening or changing. Indications for furthertesting also include presence of alarm symptoms, onset of symptoms afterage 50, and a family history of colon cancer. Tests may includecolonoscopy, computed tomography of the abdomen and pelvis, and bariumstudies of the small or large intestine.

R. Sjögren's Syndrome

The compounds and methods of this invention may be used for treatingpatients with SS. Primary Sjögren's syndrome (SS) is a chronic, slowlyprogressive, systemic autoimmune disease, which affects predominantlymiddle-aged women (female-to-male ratio 9:1), although it can be seen inall ages including childhood (Jonsson et al., 2002). It is characterizedby lymphocytic infiltration and destruction of the exocrine glands,which are infiltrated by mononuclear cells including CD4+, CD8+lymphocytes and B-cells (Jonsson et al., 2002). In addition,extraglandular (systemic) manifestations are seen in one-third ofpatients (Jonsson et al., 2001).

The glandular lymphocytic infiltration is a progressive feature (Jonssonet al., 1993), which, when extensive, may replace large portions of theorgans. Interestingly, the glandular infiltrates in some patientsclosely resemble ectopic lymphoid microstructures in the salivary glands(denoted as ectopic germinal centers) (Salomonsson et al., 2002; Xanthouet al., 2001). In SS, ectopic GCs are defined as T and B cell aggregatesof proliferating cells with a network of follicular dendritic cells andactivated endothelial cells. These GC-like structures formed within thetarget tissue also portray functional properties with production ofautoantibodies (anti-Ro/SSA and anti-La/SSB) (Salomonsson and Jonsson,2003).

In other systemic autoimmune diseases, such as RA, factors critical forectopic GCs have been identified. Rheumatoid synovial tissues with GCswere shown to produce chemokines CXCL13, CCL21 and lymphotoxin (LT)-β(detected on follicular center and mantle zone B cells). Multivariateregression analysis of these analytes identified CXCL13 and LT-β as thesolitary cytokines predicting GCs in rheumatoid synovitis (Weyand andGoronzy, 2003). Recently CXCL13 and CXCR5 in salivary glands has beenshown to play an essential role in the inflammatory process byrecruiting B and T cells, therefore contributing to lymphoid neogenesisand ectopic GC formation in SS (Salomonsson et al., 2002).

S. Psoriasis

The compounds and methods of this invention may be used for treatingpatients with psoriasis. Psoriasis is a chronic skin disease of scalingand inflammation that affects 2 to 2.6 percent of the United Statespopulation, or between 5.8 and 7.5 million people. Although the diseaseoccurs in all age groups, it primarily affects adults. It appears aboutequally in males and females. Psoriasis occurs when skin cells quicklyrise from their origin below the surface of the skin and pile up on thesurface before they have a chance to mature. Usually this movement (alsocalled turnover) takes about a month, but in psoriasis it may occur inonly a few days. In its typical form, psoriasis results in patches ofthick, red (inflamed) skin covered with silvery scales. These patches,which are sometimes referred to as plaques, usually itch or feel sore.They most often occur on the elbows, knees, other parts of the legs,scalp, lower back, face, palms, and soles of the feet, but they canoccur on skin anywhere on the body. The disease may also affect thefingernails, the toenails, and the soft tissues of the genitals andinside the mouth. While it is not unusual for the skin around affectedjoints to crack, approximately 1 million people with psoriasisexperience joint inflammation that produces symptoms of arthritis. Thiscondition is called psoriatic arthritis.

Psoriasis is a skin disorder driven by the immune system, especiallyinvolving a type of white blood cell called a T cell. Normally, T cellshelp protect the body against infection and disease. In the case ofpsoriasis, T cells are put into action by mistake and become so activethat they trigger other immune responses, which lead to inflammation andto rapid turnover of skin cells. In about one-third of the cases, thereis a family history of psoriasis. Researchers have studied a largenumber of families affected by psoriasis and identified genes linked tothe disease. People with psoriasis may notice that there are times whentheir skin worsens, then improves. Conditions that may cause flareupsinclude infections, stress, and changes in climate that dry the skin.Also, certain medicines, including lithium and beta blockers, which areprescribed for high blood pressure, may trigger an outbreak or worsenthe disease.

T. Infectious Diseases

Compounds of the present disclosure may be useful in the treatment ofinfectious diseases, including viral and bacterial infections. As notedabove, such infections may be associated with severe localized orsystemic inflammatory responses. For example, influenza may cause severeinflammation of the lung and bacterial infection can cause the systemichyperinflammatory response, including the excessive production ofmultiple inflammatory cytokines, that is the hallmark of sepsis. Inaddition, compounds of the invention may be useful in directlyinhibiting the replication of viral pathogens. Previous studies havedemonstrated that related compounds such as CDDO can inhibit thereplication of HIV in macrophages (Vazquez et al., 2005). Other studieshave indicated that inhibition of NF-kappa B signaling may inhibitinfluenza virus replication, and that cyclopentenone prostaglandins mayinhibit viral replication (e.g., Mazur et al., 2007; Pica et al., 2000).

V. PHARMACEUTICAL FORMULATIONS AND ROUTES OF ADMINISTRATION

The compounds of the present disclosure may be administered by a varietyof methods, e.g., orally or by injection (e.g. subcutaneous,intravenous, intraperitoneal, etc.). Depending on the route ofadministration, the active compounds may be coated in a material toprotect the compound from the action of acids and other naturalconditions which may inactivate the compound. They may also beadministered by continuous perfusion/infusion of a disease or woundsite.

To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a patientin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., 1984).

The therapeutic compound may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (such as, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient.

The therapeutic compound may also be administered topically to the skin,eye, or mucosa. Alternatively, if local delivery to the lungs is desiredthe therapeutic compound may be administered by inhalation in adry-powder or aerosol formulation.

Active compounds are administered at a therapeutically effective dosagesufficient to treat a condition associated with a condition in apatient. A “therapeutically effective amount” preferably reduces theamount of symptoms of the condition in the infected patient by at leastabout 20%, more preferably by at least about 40%, even more preferablyby at least about 60%, and still more preferably by at least about 80%relative to untreated subjects. For example, the efficacy of a compoundcan be evaluated in an animal model system that may be predictive ofefficacy in treating the disease in humans, such as the model systemsshown in the examples and drawings.

The actual dosage amount of a compound of the present disclosure orcomposition comprising a compound of the present disclosure administeredto a subject may be determined by physical and physiological factorssuch as age, sex, body weight, severity of condition, the type ofdisease being treated, previous or concurrent therapeutic interventions,idiopathy of the subject and on the route of administration. Thesefactors may be determined by a skilled artisan. The practitionerresponsible for administration will typically determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject. The dosage may be adjusted by theindividual physician in the event of any complication.

An effective amount typically will vary from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, fromabout 10.0 mg/kg to about 150 mg/kg in one or more dose administrationsdaily, for one or several days (depending of course of the mode ofadministration and the factors discussed above). Other suitable doseranges include 1 mg to 10000 mg per day, 100 mg to 10000 mg per day, 500mg to 10000 mg per day, and 500 mg to 1000 mg per day. In someparticular embodiments, the amount is less than 10,000 mg per day with arange of 750 mg to 9000 mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 25 mg/kg/day or less than 10 mg/kg/day. It mayalternatively be in the range of 1 mg/kg/day to 200 mg/kg/day. Forexample, regarding treatment of diabetic patients, the unit dosage maybe an amount that reduces blood glucose by at least 40% as compared toan untreated subject. In another embodiment, the unit dosage is anamount that reduces blood glucose to a level that is ±10% of the bloodglucose level of a non-diabetic subject.

In other non-limiting examples, a dose may also comprise from about 1micro-gram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milli-gram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a pharmaceutical composition of the presentdisclosure may comprise, for example, at least about 0.1% of a compoundof the present disclosure. In other embodiments, the compound of thepresent disclosure may comprise between about 2% to about 75% of theweight of the unit, or between about 25% to about 60%, for example, andany range derivable therein.

Single or multiple doses of the agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, subjects may be administered two dosesdaily at approximately 12 hour intervals. In some embodiments, the agentis administered once a day.

The agent(s) may be administered on a routine schedule. As used herein aroutine schedule refers to a predetermined designated period of time.The routine schedule may encompass periods of time which are identicalor which differ in length, as long as the schedule is predetermined. Forinstance, the routine schedule may involve administration twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between. Alternatively, the predetermined routineschedule may involve administration on a twice daily basis for the firstweek, followed by a daily basis for several months, etc. In otherembodiments, the invention provides that the agent(s) may taken orallyand that the timing of which is or is not dependent upon food intake.Thus, for example, the agent can be taken every morning and/or everyevening, regardless of when the subject has eaten or will eat.

VI. COMBINATION THERAPY

In addition to being used as a monotherapy, the compounds of the presentdisclosure may also find use in combination therapies. Effectivecombination therapy may be achieved with a single composition orpharmacological formulation that includes both agents, or with twodistinct compositions or formulations, at the same time, wherein onecomposition includes a compound of the present disclosure, and the otherincludes the second agent(s). Alternatively, the therapy may precede orfollow the other agent treatment by intervals ranging from minutes tomonths.

Various combinations may be employed, such as when a compound of thepresent disclosure is “A” and “B” represents a secondary agent,non-limiting examples of which are described below:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the compounds of the present disclosure to a patientwill follow general protocols for the administration of pharmaceuticals,taking into account the toxicity, if any, of the drug. It is expectedthat the treatment cycles would be repeated as necessary.

Beta interferons may be suitable secondary agents. These are medicationsderived from human cytokines which help regulate the immune system. Theyinclude interferon β-1b and interferon β-1a. Betaseron has been approvedby the FDA for relapsing forms of secondary progressive MS. Furthermore,the FDA has approved the use of several β-interferons as treatments forpeople who have experienced a single attack that suggests multiplesclerosis, and who may be at risk of future attacks and developingdefinite MS. For example, risk of MS may be suggested when an MRI scanof the brain shows lesions that predict a high risk of conversion todefinite MS.

Glatiramer acetate is a further example of a secondary agent that may beused in a combination treatment. Glatiramer is presently used to treatrelapsing remitting MS. It is made of four amino acids that are found inmyelin. This drug is reported to stimulate T cells in the body's immunesystem to change from harmful, pro-inflammatory agents to beneficial,anti-inflammatory agents that work to reduce inflammation at lesionsites.

Another potential secondary agent is mitoxantrone, a chemotherapy drugused for many cancers. This drug is also FDA-approved for treatment ofaggressive forms of relapsing remitting MS, as well as certain forms ofprogressive MS. It is given intravenously, typically every three months.This medication is effective, but is limited by cardiac toxicity.Novantrone has been approved by the FDA for secondary progressive,progressive-relapsing, and worsening relapsing-remitting MS.

Another potential secondary agent is natalizumab. In general,natalizumab works by blocking the attachment of immune cells to brainblood vessels, which is a necessary step for immune cells to cross intothe brain, thus reducing the immune cells' inflammatory action on brainneurons. Natalizumab has been shown to significantly reduce thefrequency of attacks in people with relapsing MS.

In the case of relapsing remitting MS, patients may be given intravenouscorticosteroids, such as methylprednisolone, as a secondary agent, toend the attack sooner and leave fewer lasting deficits.

Other common drugs for MS that may be used in combination with compoundsof the present disclosure include immunosuppressive drugs such asazathioprine, cladribine, and cyclophosphamide.

It is contemplated that other anti-inflammatory agents may be used inconjunction with the treatments of the current invention. Other COXinhibitors may be used, including arylcarboxylic acids (salicylic acid,acetylsalicylic acid, diflunisal, choline magnesium trisalicylate,salicylate, benorylate, flufenamic acid, mefenamic acid, meclofenamicacid and triflumic acid), arylalkanoic acids (diclofenac, fenclofenac,alclofenac, fentiazac, ibuprofen, flurbiprofen, ketoprofen, naproxen,fenoprofen, fenbufen, suprofen, indoprofen, tiaprofenic acid,benoxaprofen, pirprofen, tolmetin, zomepirac, clopinac, indomethacin andsulindac) and enolic acids (phenylbutazone, oxyphenbutazone,azapropazone, feprazone, piroxicam, and isoxicam. See, e.g., U.S. Pat.No. 6,025,395.

Histamine H2 receptor blocking agents may also be used in conjunctionwith the compounds of the current invention, including cimetidine,ranitidine, famotidine and nizatidine.

Treatment with acetylcholinesterase inhibitors such as tacrine,donepizil, metrifonate and rivastigmine for the treatment of Alzheimer'sand other disease in conjunction with the compounds of the presentdisclosure is contemplated. Other acetylcholinesterase inhibitors may bedeveloped which may be used once approved include rivastigmine andmetrifonate. Acetylcholinesterase inhibitors increase the amount ofneurotransmitter acetylcholine at the nerve terminal by decreasing itsbreakdown by the enzyme cholinesterase.

MAO-B inhibitors such as selegilene may be used in conjunction with thecompounds of the current invention. Selegilene is used for Parkinson'sdisease and irreversibly inhibits monoamine oxidase type B (MAO-B).Monoamine oxidase is an enzyme that inactivates the monoamineneurotransmitters norepinephrine, serotonin and dopamine.

Dietary and nutritional supplements with reported benefits for treatmentor prevention of Parkinson's, Alzheimer's, multiple sclerosis,amyotrophic lateral sclerosis, rheumatoid arthritis, inflammatory boweldisease, and all other diseases whose pathogenesis is believed toinvolve excessive production of either nitric oxide (NO) orprostaglandins, such as acetyl-L-carnitine, octacosanol, eveningprimrose oil, vitamin B6, tyrosine, phenylalanine, vitamin C, L-dopa, ora combination of several antioxidants may be used in conjunction withthe compounds of the current invention.

For the treatment or prevention of cancer, compounds of the inventionmay be combined with one or more of the following: radiation,chemotherapy agents (e.g., cytotoxic agents such as anthracyclines,vincristine, vinblastin, microtubule-targeting agents such as paclitaxeland docetaxel, 5-FU and related agents, cisplatin and otherplatinum-containing compounds, irinotecan and topotecan, gemcitabine,temozolomide, etc.), targeted therapies (e.g., imatinib, bortezomib,bevacizumab, rituximab), or vaccine therapies designed to promote anenhanced immune response targeting cancer cells.

For the treatment or prevention of autoimmune disease, compounds of theinvention may be combined with one or more of the following:corticosteroids, methotrexate, anti-TNF antibodies, other TNF-targetingprotein therapies, and NSAIDs. For the treatment of prevention ofcardiovascular diseases, compounds of the invention may be combined withantithrombotic therapies, anticholesterol therapies such as statins(e.g., atorvastatin), and surgical interventions such as stenting orcoronary artery bypass grafting. For the treatment of osteoporosis,compounds of the invention may be combined with antiresorptive agentssuch as bisphosphonates or anabolic therapies such as teriparatide orparathyroid hormone. For the treatment of neuropsychiatric conditions,compounds of the invention may be combined with antidepressants (e.g.,imipramine or SSRIs such as fluoxetine), antipsychotic agents (e.g.,olanzapine, sertindole, risperidone), mood stabilizers (e.g., lithium,valproate semisodium), or other standard agents such as anxiolyticagents. For the treatment of neurological disorders, compounds of theinvention may be combined with anticonvulsant agents (e.g., valproatesemisodium, gabapentin, phenyloin, carbamazepine, and topiramate),antithrombotic agents (e.g., tissue plasminogen activator), oranalgesics (e.g., opioids, sodium channel blockers, and otherantinociceptive agents).

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Methods and Materials

Nitric Oxide Production and Cell Viability.

RAW264.7 macrophages were pre-treated with DMSO or drugs for 2 hours,then treated with recombinant mouse IFNγ (Sigma) for 24 hours. NOconcentration in media was determined using the Griess reagent system(Promega). Cell viability was determined using WST-1 reagent (Roche).

iNOS Induction qPCR.

RAW264.7 mouse macrophage cells were pre-treated for 2 hours withcompounds at the indicated concentrations and subsequently stimulatedwith 10 ng/ml IFNγ for an additional 2 hours. mRNA levels of iNOS werequantified by qPCR and are shown relative to the vehicle-treatedIFNγ-stimulated sample which was normalized to a value of 1. Values areaverages of duplicate PCR reactions, each with triplicate wells.

iNOS Induction Western Blot.

RAW264.7 cells were pre-treated for 2 hours with indicated compounds andsubsequently stimulated with 10 ng/ml IFNγ for an additional 24 hours.iNOS protein levels were assayed by immunoblotting. Actin was used as aloading control.

Comparison Compounds.

In some of the experiments (e.g., FIGS. 17, 18), the compounds of thisinvention were compared with other synthetic triterpenoids and naturalproducts, such as those shown here:

Compound 402 can be prepared according to the methods taught by Honda etal. (1998), Honda et al. (2000b), Honda et al. (2002), Yates et al.(2007), and U.S. Pat. Nos. 6,326,507 and 6,974,801, which areincorporated herein by reference.

Example 2 Synthesis of Certain Natural Products Including anAnti-Inflammatory Pharmacore

Curcumin analogs C0008, C0009 and C0010 were synthesized as described inSchemes 1-3.

Reagents and conditions for Scheme 1: (a) 130° C., 16 h, 68%; (b) (i)LHMDS, MeI, −78° C. to rt, 3 h; (ii) LHMDS, MeI, −78° C. to rt, 3 h,71%; (c) I₂, 50° C., 24 h, 95%; (d) NaBH₄, CeCl.7H₂O, 0° C., 1.5 h, 80%;(e) TMSCl, imidazole, 0° C., 1 h, 95%; (f) (i) DIBAL-H, −78° C., 1 h;(ii) DMP, rt, 20 min, 85%.

As shown in Scheme 1, compound 3 (68%) was prepared by Diels-Alderreaction of compound 1 and 2 using a protocol modified from the methodreported by Danishefsky et al., 1979. After introduction of thegem-dimethyl group to give compound 4 (71% yield), vinyl iodide 5 (95%yield) was produced. The enone was reduced to allylic acohol 6 andprotected to give TMS ether 7 (80% for two steps). The methyl ester 7was then transformed to aldehyde 9 in two steps (91% yield).

Reagents and conditions for Scheme 2: (a) LDA, −78° C. to 0° C., 2.5 h,70%; (b) Et₃N, MsCl, 0° C., 30 min, 81%; (c) (i) ethylene glycol, TsOH,110° C., 4 h; (ii) TBSCl, imidazole, rt 43%; (d) BuLi, dimethylmethylphosphonate, −78° C. to room temperature, 91%.

Compound 12 (70%) was obtained from the aldol condensation of ketone 10and aldehyde 11 (Cardona et al., 1986) (Scheme 2). Compound 12 wastransformed to the mesylate, which eliminated in situ to give compound13 (81% yield). After protecting the enone as a ketal (43%), the methylester was transformed to dimethylphosphate 15 in excellent yield (91%).

Compound 15 and compound 9 obtained above were condensed and transformedto the target curcumin analogs (Scheme 3, below). TheHorner-Wadsworth-Emmons reaction between phosphate 15 and aldehyde 9 wasfirst attempted using NaHMDS as base. No reaction was observed, mostlikely due to the steric hindrance of 9. Using the protocol developed byRoush et al., 1984 (DIPEA/LiCl), the reaction went slowly to givecompound 16 in 28% yield. The vinyl iodide 16 was then treated withZn(CN)₂ catalyzed by Pd(PPh₃)₄ (Wu et al., 1999), and cyanide 17 (68%)was obtained. After removal of the TMS protecting group and oxidation,compound C0008 was obtained (76% yield from 17).

C0008 was the starting material for generating both C0009 and C0010(Scheme 3, below). When C0008 was treated with 6 N HCl (aq), the fullydeprotected compound C0009 (88%) was obtained. When C0008 was treatedwith TBAF, phenol C0010 was obtained in 63% yield.

Reagents and conditions for Scheme 3: (a) DIPEA, LiCl, 60° C., 20 h,28%; (b) Zn(CN)₂, Pd(PPh₃)₄, 80° C., 20 min, 68%; (c) TsOH, rt, 30 min,99%; (d) DMP, rt, 1 h, 77%; (e) 6 N HCl (aq), rt, 14 h, 88% (for C0009);TBAF, rt, 10 min, 63% (for C0010).

Reagents and conditions for Scheme 4: (a) (i) K₂CO₃, rt, 2 h; (ii)MOMCl, DIPEA, rt, 14 h, 94%.

The synthesis of Resveratrol analog R00141 is summarized in Scheme 4 andScheme 5, below. Starting material 3 was first treated with I₂/Py toproduce the vinyl iodide 21 (74%). The vinyl iodide was then protectedto give ketal 22, which was used directly in the next reaction. Ketal 22was transformed to aldehyde 24 in two steps and high yield (91%).Aldehyde 24 was reacted with diethyl phosphate 20 under basic conditionsto produce compound 25 in 89% yield. The vinyl iodide 25 was treatedwith Zn(CN)₂ and catalytic amount of Pd(PPh₃)₄, and cyanide 26 wasobtained in 62% yield. The ketal and MOM protection groups were removedin one step using TsOH to give R00141 (87%).

Reagents and conditions for Scheme 5: (a) I₂, rt, 16 h, 74%; (b)ethylene glycol, 110° C., 3 h; (c) (i) DIBAL-H, −78° C., 1 h; (ii) DMP,rt, 20 min, 91%; (d) t-BuOK, 0° C. to rt, 2 h, 89%; (e) Zn(CN)₂,Pd(PPh₃)₄, 80° C., 20 min, 62%; (f) TsOH, rt, 2 min, 87%.

Reagents and conditions for Scheme 6: (a) TsOH, rt, 89%; (b) (i) LHMDS,MeI, −78° C. to rt, 3 h; (ii) LHMDS, MeI, −78° C. to rt, 3 h, 48%; (b)Zn(CN)₂, Pd(PPh₃)₄, 80° C., 20 min, 42%; (f) TsOH, rt, 2 min, 93%.

R00142 was synthesized from compound 25 (Scheme 6). Compound 25 wastreated with TsOH, selectively removing the ketal group, to give enone27 (89%). The gem-dimethyl group was then introduced using LHMDS/MeI.From the reaction mixture, compound 28 was isolated in 48% yield. Amixture of compound 28 and 29 (14%) was also obtained. Using the sameprotocol described for the synthesis of R00141, R00142 was obtained fromcompound 28 in 39% yield. From the mixture of compound 28 and 29, amixture of R00142 and compound 31 (named as R00142-1) were obtained.

Reagents and conditions for Scheme 7: (a) t-BuOK, MeI, rt, 4 h, 62%; (b)H2, 10% Pd/C, rt, 16 h, 38%; (c) Imidazole, TMSCl, rt, 1 h, 84%; (d) (i)LDA, TsCN, −78° C. to 0° C., 1 h; (ii) DDQ, 80° C., 20 min, 50%; (e)TsOH, rt, 5 min, 95%; (f) DMP, rt, 2 h, 79%.

DHEA analog D0016 was synthesized from testosterone (Scheme 7).Testosterone was treated with t-BuOK/MeI (Cao et al., 2007) to introducethe gem-dimethyl group at the 4-position, giving compound 32 in 62%yield. Compound 32 was then hydrogenated to get 33 (38%). After the17-hydroxyl group was protected (84% yield), compound 34 was treatedwith LDA/TsCN (Kahne and Collum, 1981), followed by DDQ oxidation togive target compound D0016 in 50% yield.

D0017 and D0018 were synthesized from D0016 (Scheme 7). D0016 wastreated with TsOH to afford D0017 in 95% yield. D0017 was oxidized withDess-Martin periodinane to give compound D0018 (79%).

Reagents and conditions for Scheme 8: (a) MOMCl, DIPEA, DMAP, rt, 14 h,98%; (b) H₂O₂, NaOH, 4° C., 14 h, 71%; (c) NaCN, EtOH, 80° C., 24 h; (d)210° C., 40 min, 23% of D0014 (from 36) and 23% of D0015 (from 36).

Compound D0014 was prepared from compound 35 using the protocol reportedby Rasmusson et al., 1986 (Scheme 8). Compound 35 (produced fromtestosterone in 98% yield) was treated with alkaline hydrogen peroxideto give a mixture of epoxide epimers. The mixture was converted tobase-soluble dicyano compound 37, which was used in the next stepdirectly. Gentle pyrolysis of compound 37 afforded the corresponding4-cyano-steroids D0014 (23%) and D0015 (23%).

Reagents and conditions for Scheme 9: (a) (i) K₂CO₃, rt, 4 h; (ii) PCC,NaOAc, rt, 3 h, 95%; (b) (i) LDA, TsCN, −78° C. to 0° C., 1 h; (ii) DDQ,80° C., 20 min, 37%.

Hecogenin analog H0001 was synthesized from compound 38 (Scheme 9).Compound 38 was prepared from hecogenin acetate using the reportedprocedure (Barton et al., 1980). Acetate 38 was treated with base,followed by PCC oxidation to give ketone 39 in 95% yield. 39 was thenreacted with LDA/TsCN (Kahne and Collum, 1981), followed by DDQoxidation to give target compound H0001 (37%).

Reagents and conditions for Scheme 10: (a) ethylene glycol, CSA,cyclohexane, Dean-Stark, reflux, 20 h, 99%; (b) 3-methyl-2-butanone,Al(Oi-Pr)₃, toluene, reflux, 4 h, 72%; (c) KOt-Bu (1 M in THF), t-BuOH,allyl bromide, rt, 2 h, 73%; (d) LDA, THF, −78° C., 30 min; TsCN, −78°C., 30 min, 57%; (e) H₂ (1 atm), 10% Pd/C, THF, 2 h, 100%; (f) (i) DDQ,benzene, 80° C., 3 h; (ii) 1 N HCl (aq), THF, rt, 2 h, 10%.

Reagents and conditions for Scheme 11: (a) Grubbs' catalyst (2^(nd)generation), CH₂Cl₂, rt, 2 h, 99%; (b) LDA, THF, toluene, −78° C., 30min; TsCN, −78° C., 30 min, 34%; (c) DDQ, benzene, 80° C., 30 min, 18%;(d) 1 N HCl (aq), THF, H₂O, rt, 14 h, 64%.

Reagents and conditions for Scheme 12: (a) H₂ (1 atm), 5% Pd/C, THF, 2h, 98%; (b) LDA, THF, −78° C., 30 min; TsCN, −78° C., 30 min, 55%; (c)(i) 1,3-dibromo-5,5-dimethylhydantoin, DMF, 0° C., 2 h; (ii) pyridine,55° C., 21 h, 76% (d) 0.5 N HCl (aq), THF, rt, 50 h, 98%; (e)Al(Oi-Pr)₃, i-PrOH, toluene, 75° C., 20 h, 20%.

Reagents and conditions for Schemes 13a and 13b: (a) AcCl, MeOH, rt, 72h; (b) Ag₂CO₃/celite, toluene, reflux, 3 h, 66%; (c) MOM-Cl, i-Pr₂Net,CH₂Cl₂, 45° C., 14 h, 70%; (d) NaOMe, HCO₂Et, MeOH, rt, 2 h; (e)NH₂OH—HCl, EtOH, H₂O, 60° C., 14 h, 40%; (f) NaOMe, MeOH, THF, 55° C., 2h, 41%; (g) (i) 1,3-dibromo-5,5-dimethylhydantoin, DMF, rt, 3 h; (ii)pyridine, 55° C., 72 h, 80%; (h) 2 N HCl(Et₂O), CH₂Cl₂, rt, 14 h, 48%.

Example 3 Characterization of Certain Natural Products Including anAnti-Inflammatory Pharmacore

Compound 3:

A mixture of compound 1 (4.20 g, 24.4 mmol) and 2 (2.44 g, 24.4 mmol) intoluene (1 mL) was heated in a sealed vial at 130° C. for 16 h. Aftercooling to room temperature, THF (10 mL) and 0.1 N HCl (5 mL) wereadded. After stirring for 20 min, NaHCO₃ (aq) solution was added, andthe mixture was extracted with EtOAc. The combined extracts were washedwith water, dried with MgSO₄ and concentrated. The residue obtained waspurified by column chromatography (silica gel, 15% EtOAc in hexanes) togive compound 3 (2.84 g, 68%) as a colorless oil. ¹H NMR (400 MHz,CDCl₃) δ 6.89 (d, 1H, J=10.4 Hz), 5.98 (d, 1H, J=10.4 Hz), 3.75 (s, 3H),2.50 (m, 3H), 1.99 (m, 1H), 1.45 (s, 3H). The ¹H NMR spectrum is thesame as reported in the literature (Danishefsky et al., 1979).

Compound 4:

LHMDS (1.0 M in THF, 3.75 mL, 3.75 mmol) was added dropwise to asolution of compound 3 (505 mg, 3.00 mmol) in THF (20 mL) at −78° C.After stirring for 1 h, MeI (0.56 mL, 9.00 mmol) was added and thereaction mixture was stirred at room temperature for 2 h. After coolingto 0° C., NH₄Cl solution was added, and the mixture was extracted withCH₂Cl₂. The combined extracts were washed with water, dried with MgSO₄and concentrated. The crude product obtained was dissolved in THF (20mL) and cooled to −78° C. LHMDS (1.0 M in THF, 3.75 mL, 3.75 mmol) wasadded dropwise again. After stirring for 1 h at −78° C., MeI (0.56 mL,9.00 mmol) was added, and the reaction mixture was stirred at roomtemperature for 2 h. After cooling to 0° C., NH₄Cl solution was added,and the mixture was extracted with CH₂Cl₂. The combined extracts werewashed with water, dried with MgSO₄ and concentrated. The residueobtained was purified by column chromatography (silica gel, 10% EtOAc inhexanes) to give compound 4 (420 mg, 71%) as a white solid: ¹H NMR (300MHz, CDCl₃) δ 6.78 (dd, 1H, J=1.2, 10.2 Hz), 5.91 (d, 1H, J=10.2 Hz),3.74 (s, 3H), 2.54 (dd, 1H, J=1.2, 14.1 Hz), 1.79 (d, 1H, J=14.1 Hz),1.40 (s, 3H), 1.12 (s, 3H), 1.02 (s, 3H).

Compound 5:

A mixture of compound 4 (212 mg, 1.08 mmol), I₂ (824 mg, 3.24 mmol) andpyridine (1.5 mL) in CCl₄ (3 mL) was heated at 50° C. for 24 h. Aftercooling to room temperature, EtOAc (20 mL) was added. The mixture waswashed with Na₂S₂O₃ solution, 1 N HCl (aq), and water, then dried withMgSO₄. After concentration, the residue obtained was purified by columnchromatography (silica gel, 7% to 10% EtOAc in hexanes) to give compound5 (313 mg, 95%). ¹H NMR (400 MHz, CDCl₃) δ 7.59 (s, 1H), 3.76 (s, 3H),2.59 (d, 1H, J=14.4 Hz), 1.87 (d, 1H, J=14.4 Hz), 1.42 (s, 3H), 1.19 (s,3H), 1.05 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 25.0, 26.0, 27.9, 41.4,45.5, 46.9, 52.7, 103.0, 157.7, 174.6, 196.2.

Compound 6:

To a solution of compound 5 (200 mg, 0.62 mmol) in MeOH (6 mL) was addedCeCl₃.7H₂O (255 mg, 0.68 mmol) and NaBH₄ (26 mg, 0.68 mmol) at 0° C.After stirring for 1 h, NaBH₄ (26 mg, 0.68 mmol) was added again andstirred for another 30 min. Water was added to the reaction mixture, andthe mixture was extracted with EtOAc. The combined extracts were washedwith water, dried with MgSO₄, and concentrated. The residue obtained waspurified by column chromatography (silica gel, 10% to 20% EtOAc inhexanes) to give the allylic alcohol 6 (160 mg, 80%): ¹H NMR (400 MHz,CDCl₃) δ 6.51 (s, 1H), 3.77 (dd, 1H, J=1.2, 6.0 Hz), 3.69 (s, 3H), 2.34(d, 1H, J=14.4 Hz), 2.00 (d, 1H, J=6.0 Hz), 1.42 (d, 1H, J=14.0 Hz),1.26 (s, 3H), 1.07 (s, 3H), 0.84 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ19.1, 28.0, 28.6, 36.0, 44.5, 46.7, 52.3, 78.2, 107.3, 141.2, 175.8.

Compounds 8 and 9:

Imidazole (203 mg, 2.99 mmol) and TMSCl (190 μL, 1.49 mmol) was addedsuccessively to a solution of the alcohol 6 (160 mg, 0.50 mmol) inCH₂Cl₂ (3 mL) at 0° C. After stirring for 1 h, NaHCO₃ (aq) solution wasadded, and the mixture was extracted with CH₂Cl₂. The combined extractswere dried with MgSO₄ and concentrated to give compound 7 (194 mg, 95%).Compound 7 was used in the next step without further purification.

DIBAL-H (1.0 M in toluene, 0.50 mL, 0.50 mmol) was added to compound 7(194 mg, 0.47 mmol) in CH₂Cl₂ (5 mL) at −78° C. After stirring for 30min, DIBAL-H (1.0 M in toluene, 0.50 mL, 0.50 mmol) was added again andstirred for another 30 min. K—Na tartrate (aq) solution was added, andthe mixture was stirred at room temperature until clear. It wasextracted with CH₂Cl₂, and the combined extracts were washed with water,dried with MgSO₄, and concentrated. The residue obtained was purified bycolumn chromatography (silica gel, 10% EtOAc in hexanes) to give alcohol8 (150 mg, 87%) and aldehyde 9 (11 mg, 6.4%). Compound 8: ¹H NMR (300MHz, CDCl₃) δ 6.09 (s, 1H), 3.80 (s, 1H), 3.21 (m, 2H), 2.19 (t, 1H,J=5.4 Hz), 1.88 (d, 1H, J=14.4 Hz), 1.07 (d, 1H, J=14.4 Hz), 0.99 (s,3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.20 (s, 9H); ¹³C NMR (100 MHz, CDCl₃)δ 0.90, 24.8, 26.3, 27.7, 37.0, 37.6, 43.0, 71.2, 83.0, 100.5, 144.2.

NaHCO₃ (430 mg, 5.12 mmol) and Dess-Martin peroidinane (434 mg, 1.02mmol) in CH₂Cl₂ (2 mL) were stirred at room temperature for 20 min, andthen a solution of alcohol 8 (150 mg, 0.41 mmol) in CH₂Cl₂ (20 mL) wasadded. After stirring for 2 h, Na₂S₂O₃ (aq) solution was added andstirred for 10 min. The mixture was extracted with hexanes, and thecombined extracts were washed with NaHCO₃ (aq) solution, dried withMgSO₄, and concentrated. The residue obtained was purified by columnchromatography (silica gel, 5% EtOAc in hexanes) to give aldehyde 9 (128mg, 85%): ¹H NMR (300 MHz, CDCl₃) δ 9.37 (s, 1H), 6.29 (s, 1H), 3.89 (s,1H), 2.11 (d, 1H, J=14.0 Hz), 1.29 (d, 1H, J=14.0 Hz), 1.10 (s, 3H),1.01 (s, 3H), 0.83 (s, 3H), 0.23 (s, 9H).

Compound 12:

n-BuLi (2.5 M in hexanes, 1.46 mL, 3.65 mmol) was added todiisopropylamine (0.54 mL, 3.82 mmol) in THF (2 mL) at −78° C. Afterstirring at 0° C. for 30 min, it was cooled to −78° C. again. Compound10 (500 mg, 3.47 mmol) in THF (5 mL) was then added dropwise. Afterstirring for 1 h, compound 11 (Cardona et al., 1986) (1.11 g, 4.17 mmol)was added and the resultant mixture was stirred at −78° C. for another 2h. NH₄Cl (aq) was added to quench the reaction, and the mixture wasextracted with ether. The combined extracts were washed with water anddried with MgSO₄. After concentration, the residue obtained was purifiedby column chromatography (silica gel, 10% to 20% EtOAc in hexanes) togive product 12 (1.0 g, 70%): ¹H NMR (400 MHz, CDCl₃) δ 6.73-6.89 (m,3H), 5.10 (ddd, 1H, J=3.2, 6.4, 8.4 Hz), 3.81 (s, 3H), 3.72 (s, 3H),3.20 (d, 1H, J=2.8 Hz), 2.88 (dd, 1H, J=8.8, 17.6 Hz), 2.80 (dd, 1H,J=3.6, 17.6 Hz), 1.37 (s, 3H), 1.35 (s, 3H), 0.99 (s, 9H), 0.14 (s, 6H).

Compound 13:

Et₃N (1.06 mL, 7.62 mmol) and MsCl (0.24 mL, 3.09 mmol) were addedsuccessively to a solution of compound 12 (1.00 g, 2.42 mmol) in CH₂Cl₂(20 mL) at 0° C. After stirring for 30 min, NaHCO₃ (aq) solution wasadded, and the mixture was extracted with EtOAc. The combined extractswere washed with water and dried with MgSO₄. After concentration, theresidue obtained was purified by column chromatography (silica gel, 10%EtOAc in hexanes) to give product 13 (0.78 g, 81%): ¹H NMR (400 MHz,CDCl₃) δ 7.64 (d, 1H, J=16.0 Hz), 7.06 (m, 1H), 6.99 (bs, 1H), 6.83 (d,1H, J=8.0 Hz), 6.63 (d, 1H, J=16.0 Hz), 3.84 (s, 3H), 3.71 (s, 3H), 1.43(s, 6H), 0.98 (s, 9H), 0.16 (s, 6H).

Compound 14:

A mixture of compound 13 (3.70 g, 9.44 mmol), ethylene glycol (6.0 mL),and TsOH (0.80 g) in toluene (25 mL) was refluxed with a Dean-Starkapparatus for 4 h. After cooling to room temperature, the mixture waswashed with NaHCO₃ (aq) solution and water, then dried over MgSO₄. Afterconcentration, the brown oil was dissolved in DMF (5 mL). Imidazole(2.70 g, 39.7 mmol) and TBSCl (1.50 g, 10 mmol) were added. Afterstirring for 30 min, TBSCl (500 mg, 3.33 mmol) was added again andstirred for an additional 30 min. NaHCO₃ (aq) solution was then addedand the mixture was extracted with EtOAc. The combined extracts werewashed with water and dried with MgSO₄. After concentration, the residueobtained was purified by column chromatography (silica gel, 10% to 15%EtOAc in hexanes) to give product 14 (1.71 g, 43%): ¹H NMR (400 MHz,CDCl₃) δ 6.85-6.90 (m, 2H), 6.79 (d, 1H, J=8.4 Hz), 6.60 (d, 1H, J=16.0Hz), 6.07 (d, 1H, J=16.0 Hz), 3.95 (m, 4H), 3.83 (s, 3H), 3.70 (s, 3H),1.30 (s, 6H), 1.00 (s, 9H), 0.15 (s, 6H).

Compound 15:

n-BuLi (2.5 M in hexanes, 3.30 mL, 8.25 mmol) was added to dimethylmethylphosphonate (1.0 mL, 9.35 mmol) in THF (10 mL) at −78° C. Afterstirring for 15 min, a solution of compound 14 (1.20 g, 2.75 mmol) inTHF (5 mL) was added dropwise. After stirring at room temperature for 2h, NH₄Cl (aq) solution was added. The mixture was extracted with EtOAcand the combined extracts were washed with water and dried with MgSO₄.After concentration, the residue obtained was purified by columnchromatography (silica gel, 50% to 100% EtOAc in hexanes) to giveproduct 14 (1.32 g, 91%): ¹H NMR (400 MHz, CDCl₃) δ 6.84-6.89 (d, 2H),6.79 (d, 1H, J=8.0 Hz), 5.57 (d, 1H, J=16.0 Hz), 5.87 (d, 1H, J=16.0Hz), 3.90-4.01 (m, 4H), 3.82 (s, 3H), 3.80 (s, 3H), 3.77 (s, 3H), 3.47(d, 2H, J=20.4 Hz), 1.26 (s, 6H), 0.99 (s, 9H), 0.15 (s, 6H).

Compound 16:

The condensation between compound 9 and 15 was performed using theprocedure developed by Roush et al., 1984. MeCN (10 mL) and DIPEA (1.25mL, 7.18 mmol) were added to a mixture of LiCl (121 mg, 2.85 mmol) andcompound 15 (830 mg, 1.71 mmol) to give a yellow solution. Compound 9(520 mg, 1.42 mmol) in MeCN (5 mL) was then added. The resultantreaction mixture was heated at 60° C. for 20 h and cooled to roomtemperature. EtOAc was added and the mixture was washed with water,NaHCO₃ (aq) solution, and brine, then dried with MgSO₄ and concentrated.The residue obtained was purified by column chromatography (silica gel,6% to 7% EtOAc in hexanes) to give product 16 (310 mg). The recoveredstarting materials 9 and 15 were treated again with the same reactionconditions to produce another 285 mg of product 16. The overall yield is55%: ¹H NMR (400 MHz, CDCl₃) δ 6.82-6.86 (m, 2H), 6.77 (d, 1H, J=8.0Hz), 6.70 (bs, 2H), 6.55 (d, 1H, J=15.6 Hz), 6.28 (s, 1H), 5.88 (d, 1H,J=15.6 Hz), 3.90 (m, 4H), 3.84 (bs, 1H), 3.81 (s, 3H), 1.80 (d, 1H,J=14.8 Hz), 1.40 (d, 1H, J=14.8 Hz), 1.22 (s, 6H), 1.11 (s, 3H), 0.99(s, 3H), 0.98 (s, 9H), 0.83 (s, 3H), 0.23 (s, 9H), 0.14 (s, 6H); ¹³C NMR(100 MHz, CDCl₃) δ −4.4, 1.4, 18.7, 20.7, 24.1, 25.9, 28.8, 28.9, 37.7,42.9, 44.9, 54.1, 55.7, 64.9, 65.0, 81.5, 105.4, 110.3, 111.4, 120.4,121.1, 124.1, 124.2, 130.1, 132.2, 143.3, 145.5, 151.2, 152.7, 201.7.

Compound 17:

Pd(PPh₃)₄ (90 mg, 0.078 mmol) in DMF (10 mL) was added to a mixture ofcompound 16 (595 mg, 0.77 mmol) and Zn(CN)₂ (363 mg, 3.10 mmol) at roomtemperature under Ar. The slurry was heated at 80° C. for 20 min andcooled to room temperature. Ether was added, and the mixture was washedwith water, dried with MgSO₄ and concentrated. The residue obtained waspurified by column chromatography (silica gel, 5% to 10% EtOAc inhexanes) to give product 17 (352 mg, 68%): ¹H NMR (400 MHz, CDCl₃) δ6.82-6.85 (m, 2H), 6.79 (d, 1H, J=8.0 Hz), 6.68 (m, 2H), 6.55 (d, 1H,J=16.0 Hz), 6.51 (bs, 1H), 5.86 (d, 1H, J=16.0 Hz), 3.90 (m, 5H), 3.82(s, 3H), 1.80 (d, 1H, J=14.4 Hz), 1.42 (d, 1H, J=14.0 Hz), 1.23 (s, 3H),1.23 (s, 3H), 1.15 (s, 3H), 0.99 (s, 9H), 0.95 (s, 3H), 0.78 (s, 3H),0.23 (s, 9H), 0.15 (s, 6H).

Compound 18:

TsOH (240 mg, 1.26 mmol) was added to a solution of compound 17 (160 mg,0.24 mmol) in acetone (5 mL) and water (1 mL) at room temperature. Afterstirring for 30 min, NaHCO₃ (aq) solution was added, and the mixture wasextracted with EtOAc. The combined extracts were washed with water anddried with MgSO₄. After concentration, the residue obtained was purifiedby column chromatography (silica gel, 20% to 33% EtOAc in hexanes) togive product 18 (142 mg, 99%): ¹H NMR (400 MHz, CDCl₃) δ 6.82-6.85 (m,2H), 6.79 (d, 1H, J=8.0 Hz), 6.77 (d, 1H, J=16.0 Hz), 6.66 (d, 1H,J=16.0 Hz), 6.59 (m, 1H), 6.55 (d, 1H, J=15.6 Hz), 5.85 (d, 1H, J=15.6Hz), 3.96 (dd, 1H, J=1.6, 6.8 Hz), 3.92 (m, 4H), 3.82 (s, 3H), 1.82 (d,1H, J=14.4 Hz), 1.48 (d, 1H, J=14.0 Hz), 1.24 (s, 3H), 1.23 (s, 3H),1.15 (s, 3H), 1.03 (s, 3H), 0.99 (s, 9H), 0.80 (s, 3H), 0.15 (s, 6H).The compound was contaminated with some unidentified impurities.

Compound C0008:

Using the procedure described for the synthesis of compound 9 fromcompound 8, C0008 (110 mg, 77%) was produced from compound 18 (142 mg,0.24 mmol): ¹H NMR (300 MHz, CDCl₃) δ 7.50 (d, 1H, J=1.5 Hz), 6.76-6.83(m, 4H), 6.72 (d, 1H, J=16.2 Hz), 6.53 (d, 1H, J=15.9 Hz), 5.82 (d, 1H,J=15.9 Hz), 3.91 (m, 4H), 3.81 (s, 3H), 2.06 (dd, 1H, J=1.5, 14.7 Hz),1.95 (d, 1H, J=14.4 Hz), 1.33 (s, 3H), 1.24 (s, 3H), 1.24 (s, 3H), 1.16(s, 3H), 1.05 (s, 3H), 0.98 (s, 9H), 0.14 (s, 6H); m/z 616.3 (M+Na⁺).

Compound C0009:

A mixture of C0008 (90 mg, 0.15 mmol), 6 N HCl (aq) (1 mL) and THF (5mL) was stirred at room temperature overnight. EtOAc was added, and themixture was washed with water, dried with MgSO₄, and concentrated. Theresidue obtained was purified by column chromatography (silica gel, 33%EtOAc in hexanes) to give product C0009 (58 mg, 88%): ¹H NMR (300 MHz,CDCl₃) δ 7.64 (d, 1H, J=15.3 Hz), 7.39 (d, 1H, J=1.5 Hz), 7.10 (dd, 1H,J=1.8, 8.4 Hz), 6.89-6.98 (m, 3H), 6.56 (d, 1H, J=15.3 Hz), 6.12 (d, 1H,J=15.3 Hz), 5.93 (s, 1H), 3.94 (s, 3H), 2.03 (dd, 1H, J=1.8, 14.7 Hz),1.92 (d, 1H, J=14.4 Hz), 1.42 (s, 3H), 1.41 (s, 3H), 1.33 (s, 3H), 1.11(s, 3H), 0.92 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 20.8, 20.8, 25.8,25.9, 29.2, 39.4, 41.5, 47.0, 56.0, 60.4, 110.3, 114.0, 114.9, 115.7,118.3, 123.4, 124.0, 126.4, 145.2, 146.8, 148.7, 151.1, 162.5, 196.2,197.4, 197.6; m/z 458.1 (M+Na⁺).

Compound C0010:

TBAF (1.0 M in THF, 74 μL, 0.074 mmol) was added to a solution of C0008(40 mg, 0.067 mmol) in THF (3 mL). After stirring for 10 min, EtOAc wasadded. The mixture was washed with water, dried with MgSO₄, andconcentrated. The residue obtained was purified by column chromatography(silica gel, 33% EtOAc in hexanes) to give product C0010 (20 mg, 63%):¹H NMR (400 MHz, CDCl₃) δ 7.49 (d, 1H, J=1.6 Hz), 6.86 (m, 3H), 6.84 (d,1H, J=16.0 Hz), 6.72 (d, 1H, J=16.0 Hz), 6.53 (d, 1H, J=16.0 Hz), 5.82(d, 1H, J=16.0 Hz), 5.71 (s, 1H), 3.92 (bs, 7H), 2.06 (dd, 1H, J=2.0,14.4 Hz), 1.95 (d, 1H, J=14.4 Hz), 1.34 (s, 3H), 1.25 (s, 3H), 1.24 (s,3H), 1.17 (s, 3H), 1.06 (s, 3H); m/z 480.2 (M+1).

Compound 20:

A mixture of K₂CO₃ (7.00 g, 50.7 mmol) and compound 19 (2.70 g, 9.44mmol) in MeOH (150 mL) was stirred at room temperature for 2 h. Themixture was filtered, and the filtrate obtained was concentrated to givethe crude potassium salt, which was suspended in THF (50 mL). DIPEA(5.42 mL, 31.2 mmol) and MOMCl (2.70 mL, 35.5 mmol) were addedsuccessively. The white slurry was stirred at room temperatureovernight. EtOAc was added, and the mixture was washed with water, driedwith MgSO₄, and concentrated. The oil obtained was purified by columnchromatography (silica gel, 5% MeOH in CH₂Cl₂) to give product 20 (2.53g, 94%).

Compound 21:

A mixture of compound 3 (280 mg, 1.67 mmol), I₂ (630 mg, 2.48 mmol), andpyridine (2 mL) in CCl₄ (4 mL) was stirred at room temperature for 16 h.EtOAc was added, and the mixture was washed with Na₂S₂O₃ (aq) solution,1 N HCl (aq), and water, then dried with MgSO₄. After concentration, theresidue obtained was purified by column chromatography (silica gel, 12%EtOAc in hexanes) to give compound 21 (364 mg, 74%): ¹H NMR (300 MHz,CDCl₃) δ 7.68 (s, 1H), 3.77 (s, 3H), 2.69-2.74 (m, 2H), 2.52 (m, 1H),2.04 (m, 1H), 1.47 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 24.7, 32.5, 33.4,48.0, 52.8, 104.3, 159.6, 173.3, 191.1.

Compound 22:

A mixture of compound 21 (370 mg, 1.26 mmol), ethylene glycol (1 mL),PPTS (80 mg) in toluene (3 mL) was heated at reflux with a Dean-Starkapparatus for 3 h. After cooling to room temperature, EtOAc was added.The mixture was washed with NaHCO₃ (aq) solution, water, and dried withMgSO₄. After concentration, crude product 22 (400 mg, 94%) was obtained,which was used in the next step without further purification. Compound22: ¹H NMR (300 MHz, CDCl₃) δ 6.65 (s, 1H), 4.21 (m, 2H), 3.98 (m, 2H),3.69 (s, 3H), 2.30 (m, 1H), 1.96 (m, 2H), 1.75 (m, 1H), 1.30 (s, 3H).

Compounds 23, 24:

Using the procedure described for the synthesis of compounds 8 and 9from compound 7, compound 23 (185 mg, 50%) and 24 (150 mg, 41%) wasproduced from compound 22 (400 mg, 1.18 mmol).

Using the procedure described for the synthesis of compound 9 fromcompound 8, compound 24 (158 mg, 85%) was produced from compound 23 (185mg, 0.60 mmol).

Compound 23:

¹H NMR (400 MHz, CDCl₃) δ 6.45 (s, 1H), 4.21 (m, 2H), 3.98 (m, 2H), 3.39(m, 2H), 1.85-2.00 (m, 4H), 1.54 (m, 1H), 1.04 (s, 3H); ¹³C NMR (100MHz, CDCl₃) δ 22.2, 28.7, 30.5, 42.4, 65.4, 65.6, 69.7, 104.5, 105.8,149.6.

Compound 24:

¹H NMR (400 MHz, CDCl₃) δ 9.43 (s, 1H), 6.47 (s, 1H), 4.21 (m, 2H), 3.98(m, 2H), 2.16 (m, 1H), 1.94 (m, 2H), 1.71 (m, 1H), 1.16 (s, 3H); ¹³C NMR(100 MHz, CDCl₃) δ 20.8, 27.3, 30.6, 52.1, 65.7, 65.8, 105.1, 107.4,143.4, 199.9.

Compound 25:

Potassium t-butoxide (790 mg, 7.05 mmol) in THF (20 mL) was added to asolution of compound 20 (2.13 g, 7.39 mmol) in THF (10 mL) at 0° C.After stirring for 45 min, a solution of compound 24 (512 mg, 1.67 mmol)in THF (10 mL) was added. The mixture was stirred at 0° C. for 1 h andat room temperature for another 1 h. EtOAc was added, and the mixturewas washed with water and dried with MgSO₄. After concentration, theresidue obtained was purified by column chromatography (silica gel, 12%EtOAc in hexanes) to give product 25 (680 mg, 89%): ¹H NMR (400 MHz,CDCl₃) δ 7.27 (d, 2H, J=10.4 Hz), 6.97 (d, 2H, J=10.4 Hz), 6.47 (s, 1H),6.27 (d, 1H, J=16.4 Hz), 5.92 (d, 1H, J=16.0 Hz), 5.16 (s, 2H), 4.22 (m,2H), 3.97 (m, 2H), 3.48 (s, 3H), 1.94 (m, 2H), 1.80 (m, 2H), 1.12 (s,3H); NMR (100 MHz, CDCl₃) δ 27.2, 30.6, 33.3, 43.0, 55.9, 65.4, 65.8,94.3, 103.4, 105.9, 116.2, 127.3, 128.0, 130.9, 133.7, 150.4, 156.6.

Compound 26:

Using the procedure described for the synthesis of compound 17 fromcompound 16, compound 26 (36 mg, 62%) was produced from compound 25 (75mg, 0.17 mmol). ¹H NMR (400 MHz, CDCl₃) δ 7.28 (d, 2H, J=10.4 Hz), 6.98(d, 2H, J=10.4 Hz), 6.54 (s, 1H), 6.21 (d, 1H, J=16.4 Hz), 5.91 (d, 1H,J=16.0 Hz), 5.17 (s, 2H), 4.24 (m, 2H), 3.96-4.07 (m, 2H), 3.47 (s, 3H),1.78-1.89 (m, 4H), 1.27 (s, 3H).

Compound R00141:

TsOH (77 mg, 0.40 mmol) was added to a solution of compound 26 (36 mg,0.081 mmol) in acetone (3 mL) at room temperature, and acetone wasremoved under reduced pressure to give a white solid, which was driedunder vacuum (2 mm Hg) for 2 min. Acetone (10 mL) and NaHCO₃ (1.0 g)were added. After stirring for 5 min, the mixture was filtered through apad of celite. The filtrate was concentrated and the crude product waspurified by column chromatography (silica gel, 15% to 25% EtOAc inhexanes) to give product R00141 (18 mg, 87%): ¹H NMR (400 MHz, CDCl₃) δ7.47 (d, 1H, J=1.2 Hz), 7.25 (d, 2H, J=9.2 Hz), 6.82 (d, 2H, J=8.8 Hz),6.27 (d, 1H, J=16.4 Hz), 5.96 (d, 1H, J=16.4 Hz), 5.26 (bs, 1H),2.52-2.66 (m, 2H), 2.04-2.14 (m, 2H), 1.43 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 26.8, 33.6, 34.2, 40.0, 114.0, 115.7, 116.4, 127.8, 128.6,129.2, 130.5, 155.8, 167.6, 192.1.

Compound 27:

TsOH (5.04 g, 26.5 mmol) was added to a solution of compound 25 (680 mg,1.50 mmol) in acetone (25 mL) and water (5 mL). The mixture was stirredat room temperature until compound 25 was completely consumed by TLCanalysis. EtOAc was added, and the mixture was washed with water, NaHCO₃(aq) solution, and dried with MgSO₄. After concentration, the residueobtained was purified by column chromatography (silica gel, 10% EtOAc inhexanes) to give product 25 (550 mg, 89%): ¹H NMR (300 MHz, CDCl₃) δ7.52 (s, 1H), 7.28 (m, 2H), 7.00 (m, 2H), 6.33 (d, 1H, J=16.2 Hz), 5.98(d, 1H, J=16.2 Hz), 5.18 (s, 2H), 3.48 (s, 3H), 2.68 (m, 2H), 2.09 (m,2H), 1.37 (s, 3H).

Compound 28:

Using the procedure described for the synthesis of compound 4 fromcompound 3, compound 28 (280 mg, 48%) was produced from compound 27 (550mg, 1.34 mmol). From this reaction, a mixture of compound 28 and 29 (85mg, 14%) was also obtained.

Compound 28:

¹H NMR (400 MHz, CDCl₃) δ 7.61 (s, 1H), 7.28 (d, 2H, J=8.8 Hz), 6.99 (d,2H, J=8.8 Hz), 6.29 (d, 1H, J=16.4 Hz), 6.07 (d, 1H, J=16.4 Hz), 5.17(s, 2H), 3.47 (s, 3H), 2.05 (dd, 1H, J=1.2, 14.4 Hz), 1.99 (d, 1H,J=14.0 Hz), 1.32 (s, 3H), 1.20 (s, 3H), 1.16 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 27.1, 27.8, 29.9, 42.0, 43.2, 48.6, 55.9, 94.3, 102.8, 116.3,127.2, 128.0, 130.5, 134.8, 156.8, 162.6, 197.2.

Compound 30:

Using the procedure described for the synthesis of compound 17 fromcompound 16, compound 30 (90 mg, 42%) was produced from compound 28 (280mg, 0.66 mmol): ¹H NMR (400 MHz, CDCl₃) δ 7.56 (d, 1H, J=1.6 Hz), 7.28(m, 2H), 7.01 (m, 2H), 6.26 (d, 1H, J=16.4 Hz), 6.07 (d, 1H, J=16.4 Hz),5.18 (s, 2H), 3.47 (s, 3H), 2.07 (dd, 1H, J=1.6, 14.8 Hz), 1.96 (d, 1H,J=14.8 Hz), 1.39 (s, 3H), 1.20 (s, 3H), 1.17 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 26.3, 26.5, 29.7, 39.4, 41.7, 47.8, 56.0, 94.2, 114.5, 114.9,116.4, 127.3, 128.9, 130.0, 133.3, 157.1, 165.7, 197.2.

Compound C00142:

Using the procedure described for the synthesis of C00141 from compound26, compound C00142 (70 mg, 93%) was produced from compound 30 (90 mg,0.27 mmol): ¹H NMR (300 MHz, CDCl₃) δ 7.56 (d, 1H, J=1.5 Hz), 7.21 (m,2H), 6.83 (m, 2H), 6.23 (d, 1H, J=16.5 Hz), 6.07 (bs, 1H), 6.01 (d, 1H,J=16.5 Hz), 2.04 (dd, 1H, J=1.5, 14.7 Hz), 1.94 (d, 1H, J=14.7 Hz), 1.37(s, 3H), 1.19 (s, 3H), 1.16 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 26.3,26.5, 29.6, 39.4, 41.7, 47.7, 114.5, 114.7, 115.7, 127.5, 128.7, 129.1,132.5, 155.9, 166.4, 197.7; m/e 282.1 (M+1).

R00142-1:

Using the procedure described for the synthesis of C00142 from compound28, C00142-1 (a 1:1 mixture of 31 and C00142) was prepared from amixture of compound 28 and 29 (2 mg, 4.6 μmol). The 1H NMR that can beidentified for compound 31 is: (400 MHz, CDCl₃) δ 7.41 (d, 1H, J=2.4Hz), 6.23 (d, 1H, J=16.4 Hz), 5.97 (d, 1H, J=16.4 Hz), 2.61 (m, 1H),1.39 (s, 3H), 1.15 (d, 3H, J=6.8 Hz); LC-MS shows the retention time forcompound 31 and C00142 are 6.52 min with m/z 290.0 (M+Na⁺) and 7.36 minwith m/z 304.0 (M+Na⁺) respectively.

Compound 32:

Testosterone (5.19 g, 18.0 mmol) was added in one portion to a solutionof t-BuOK (5.96 g, 53.2 mmol) in t-BuOH (100 mL) at room temperature.After stirring for 5 min, MeI (6.64 mL, 106.4 mmol) was added dropwiseover 10 min. After stirring at room temperature for 4 h, water (75 mL)was added, and t-BuOH was removed under reduced pressure. The whitesolid that precipitated was collected by filtration and washed withwater. The white solid was dissolved in CH₂Cl₂, dried with MgSO₄ andconcentrated. The crude product was recrystallized from acetone (80 mL)to give compound 32 (3.50 g, 62%) as a white crystalline solid: ¹H NMR(400 MHz, CDCl₃) δ 5.56 (dd, 1H, J=2.8, 6.8 Hz), 3.66 (m, 1H), 2.40-2.62(m, 2H), 1.99-2.19 (m, 3H), 1.86 (m, 1H), 1.26-1.70 (m, 9H), 1.24 (s,6H), 0.94-1.18 (m, 3H), 0.87 (s, 3H), 0.77 (s, 3H); ¹³C NMR (75 MHz,CDCl₃) δ 11.0, 19.3, 20.8, 23.3, 27.2, 30.2, 30.5, 31.2, 31.2, 32.0,33.6, 36.5, 37.1, 42.7, 48.6, 49.0, 51.3, 81.7, 119.6, 149.8, 216.7.

Compound 33:

Compound 32 (1.05 g, 3.32 mmol) and 10% Pd on carbon (500 mg) in EtOH(50 mL) was hydrogenated (1 atm) for 16 h at room temperature. Thereaction mixture was filtered through a pad of celite, and the filtratewas concentrated to give crude product, which was purified by columnchromatography (silica gel, 5% to 9% CH₂Cl₂ in EtOAc) to give compound33 (400 mg, 38%) as white solid: ¹H NMR (300 MHz, CDCl₃) δ 3.63 (m, 1H),2.63 (ddd, 1H, J=6.6, 12.9, 15.5 Hz), 2.32 (ddd, 1H, J=3.3, 5.4, 15.5Hz), 1.92-2.14 (m, 2H), 1.77-1.83 (m, 2H), 1.19-1.65 (m, 11H), 1.08 (m,1H), 1.06 (s, 6H), 1.05 (s, 3H), 0.65-0.95 (m, 3H), 0.75 (s, 3H); ¹³CNMR (75 MHz, CDCl₃) δ 11.1, 14.0, 20.4, 21.8, 22.3, 23.3, 25.7, 30.5,32.0, 34.6, 35.2, 36.5, 36.5, 38.1, 42.9, 47.8, 50.9, 55.5, 55.8, 81.8,217.4.

Compound 34:

Imidazole (125 mg, 1.84 mmol) and TMSCl (155 μL, 1.22 mmol) were addedto a solution of compound 33 (195 mg, 0.61 mmol) in CH₂Cl₂ (6.0 mL) atroom temperature. After stirring for 1 h, the reaction mixture waswashed with NaHCO₃ (aq) solution, dried with MgSO₄ and concentrated. Theresidue obtained was purified by column chromatography (silica gel, 5%hexanes in ether) to give compound 34 (200 mg, 84%): ¹H NMR (400 MHz,CDCl₃) δ 3.53 (dd, 1H, J=8.0, 8.4 Hz), 2.63 (ddd, 1H, J=7.2, 13.2, 15.2Hz), 2.32 (ddd, 1H, J=3.2, 5.2, 15.2 Hz), 1.97 (ddd, 1H, J=3.2, 5.6,13.2 Hz), 1.71-1.91 (m, 3H), 1.19-1.59 (m, 10H), 1.06 (s, 3H), 1.05 (s,6H), 0.78-0.99 (m, 3H), 0.70 (s, 3H), 0.66 (m, 1H), 0.07 (s, 9H); ¹³CNMR (75 MHz, CDCl₃) δ 0.1, 11.3, 13.9, 20.5, 21.7, 22.3, 23.4, 25.7,30.8, 32.1, 34.7, 35.2, 36.5, 36.8, 38.1, 42.9, 47.8, 50.6, 55.6, 56.0,81.6, 217.3.

Compound D0016:

n-BuLi (2.5 M in hexanes, 0.41 mL, 1.03 mmol) was added todiisopropylamine (159 μL, 1.12 mmol) in THF (0.6 mL) at −78° C. Afterstirring at 0° C. for 30 min, the reaction mixture was cooled to −78° C.again, and compound 34 (200 mg, 0.51 mmol) in THF (2 mL) was addeddropwise. After stirring for 30 min at −78° C., TsCN (371 mg, 2.04 mmol)in THF (2.0 mL) was added. After stirring for another 30 min, water (1mL) was added to quench the reaction. The reaction mixture was adjustedto pH 3 using 3 N HCl (aq) and extracted with EtOAc. The combinedextracts were washed with water, dried with MgSO₄ and concentrated. Thecrude product obtained was dissolved in benzene (15 mL) and DDQ (116 mg,0.51 mmol) was added. The red solution was refluxed for 20 min andcooled to room temperature. The reaction mixture was washed with NaHCO₃(aq) solution and water, then dried with MgSO₄. After concentration, theresidue obtained was purified by column chromatography (silica gel, 0 to5% EtOAc in CH₂Cl₂) to give D0016 (106 mg, 50%): ¹H NMR (400 MHz, CDCl₃)δ 7.84 (s, 1H), 3.55 (dd, 1H, J=8.4, 8.4 Hz), 1.80-1.92 (m, 3H),1.64-1.73 (m, 3H), 1.40-1.58 (m, 5H), 1.26 (m, 1H), 1.19 (s, 3H), 1.17(s, 3H), 1.11 (s, 3H), 0.86-1.16 (m, 4H), 0.73 (s, 3H), 0.08 (s, 9H).

Compound D0017:

p-TsOH (220 mg, 1.16 mmol) was added to a solution of D0016 (95 mg, 0.23mmol) in acetone (2 mL) and water (0.4 mL). After stirring at roomtemperature for 5 min, NaHCO₃ (aq) solution was added, and the mixturewas extracted with CH₂Cl₂. The combined extracts were dried with MgSO₄and concentrated. The crude product obtained was purified by columnchromatography (silica gel, 33% EtOAc in hexanes) to give D0017 (75 mg,95%): ¹H NMR (300 MHz, CDCl₃) δ 7.85 (s, 1H), 3.67 (dd, 1H, J=8.4, 8.7Hz), 2.08 (m, 1H), 1.20-1.96 (m, 12H), 1.19 (s, 3H), 1.18 (s, 3H), 1.11(s, 3H), 0.86-1.14 (m, 4H), 0.78 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ11.5, 16.1, 20.7, 21.6, 22.0, 23.5, 27.0, 30.6, 31.5, 35.6, 36.5, 40.8,43.2, 45.1, 51.1, 51.3, 51.9, 81.6, 114.7, 115.3, 168.8, 198.2.

Compound D0018:

NaHCO₃ (140 mg, 1.66 mmol) and Dess-Martin periodinane (177 mg, 0.42mmol) were added successively to a solution of D0017 (57 mg, 0.17 mmol)in CH₂Cl₂ (3 mL) at room temperature. After stirring for 2 h, 5% Na₂S₂O₃(aq) solution was added. The reaction mixture was extracted with ether,and the combined extracts were washed with NaHCO₃ (aq) solution, driedwith MgSO₄ and concentrated. The crude product obtained was purified bycolumn chromatography (silica gel, 20% EtOAc in hexanes) to give D0018(45 mg, 79%) which was contaminated with some impurities.Recrystallization from EtOAc/hexanes afforded purified D0018 (38 mg) asa white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.84 (s, 3H), 2.48 (m, 1H),1.26-2.18 (m, 12H), 1.21 (s, 3H), 1.20 (s, 3H), 1.13 (s, 3H), 1.08 (m,1H), 0.91 (s, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 13.8, 15.8, 20.1, 21.2,21.6, 21.7, 26.7, 30.5, 31.0, 34.8, 35.6, 40.5, 44.8, 47.5, 50.9, 51.1,51.5, 114.6, 114.8, 167.8, 197.7, 219.9.

Compound 35:

Diisopropylethylamine (12.2 mL, 70.0 mmol), MOMCl (2.66 mL, 35.0 mmol)and DMAP (0.21 g, 1.7 mmol) were added successively to a stirredsolution of testosterone (5.05 g, 17.5 mmol) in CH₂Cl₂ (50 mL). Afterstirring at room temperature for 14 h, NaHCO₃ (aq) solution was added.After stirring for 10 min, the organic layer was separated, washed with1 N HCl (aq), NaHCO₃ (aq) solution, and water, then dried with MgSO₄.After concentration, the residue obtained was purified by columnchromatography (silica gel, 9% to 33% EtOAc in hexanes) to give compound35 (5.66 g, 98%) as a white solid: ¹H NMR (400 MHz, CDCl₃) δ 5.73 (bs,1H), 4.63 (m, 2H), 3.53 (dd, 1H, J=8.4, 8.4 Hz), 3.35 (s, 3H), 2.32-2.47(m, 3H), 2.28 (m, 1H), 2.04 (m, 2H), 1.90 (m, 1H), 1.85 (m, 1H),1.24-1.75 (m, 7H), 1.19 (s, 3H), 1.15 (m, 1H), 0.88-1.06 (m, 3H), 0.82(s, 3H).

Compounds D0014 and D0015:

A solution of compound 35 (1.66 g, 5.0 mmol) in MeOH (80 mL) was treatedwith 30% H₂O₂ (aq) (3.52 mL, 35.2 mmol) and NaOH (aq) solution (2.5 N,1.40 mL, 3.50 mmol) at 0° C. After stirring at 4° C. for 14 h, water(200 mL) was added, and the reaction mixture was extracted with EtOAc.The combined organic layer was washed with water, dried with MgSO₄ andconcentrated to give epoxide 36 (1.24 g, 71%) as mixture of epimers. Asolution of epoxide 36 (1.20 g, 3.47 mmol) in EtOH (60 mL) was treatedwith a solution of NaCN (1.70 g, 34.7 mmol) in water (18 mL). Afterrefluxing for 24 h, EtOH was removed by evaporation. 10% NaOH (aq)solution (30 mL) was added at room temperature, and the mixture wasextracted with ether. The aqueous layer was acidified with 6 N HCl (aq)(30 mL) at 0° C. to pH 2 and extracted with EtOAc. The combined EtOAcextracts were washed with water, dried with MgSO₄ and concentrated togive crude 37 as a white foam solid. Compound 37 was heated at 210° C.under vacuum (5 mmHg) for 40 min and cooled to room temperature. Thebrown viscous oil was purified by column chromatography (silica gel, 33%to 50% EtOAc in hexanes) to give compound D0014 (290 mg, 23% from 36)and D0015 (260 mg, 23% from 36) as white solids.

D0014:

¹H NMR (400 MHz, CDCl₃) δ 4.63 (m, 2H), 3.54 (dd, 1H, J=8.4 Hz), 3.35(s, 3H), 3.07 (ddd, 1H, J=2.4, 3.6, 15.2 Hz), 1.90-2.13 (m, 4H),1.43-1.80 (m, 7H), 1.30-1.40 (m, 2H), 1.27 (s, 3H), 1.10-1.21 (m, 2H),0.94-1.10 (m, 2H), 0.83 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 11.6, 18.0,20.5, 23.1, 27.9, 31.1, 32.0, 33.0, 34.0, 34.9, 36.6, 40.2, 42.4, 50.1,53.7, 55.1, 86.0, 95.9, 112.1, 114.0, 184.0, 192.3; m/z 358.2 (M+1).

D0015:

¹H NMR (300 MHz, CDCl₃) δ 3.67 (dd, 1H, J=8.4 Hz), 3.07 (ddd, 1H, J=2.7,3.9, 15.3 Hz), 2.40-2.56 (m, 3H), 1.96-2.16 (m, 3H), 1.89 (ddd, 1H,J=3.0, 3.6, 12.6 Hz), 1.57-1.79 (m, 5H), 1.43-1.55 (m, 2H), 1.34 (m,1H), 1.28 (s, 3H), 0.80-1.20 (m, 4H), 0.80 (s, 3H); ¹³C NMR (75 MHz,CDCl₃) δ 11.0, 18.0, 20.6, 23.2, 30.3, 31.2, 32.0, 33.0, 34.0, 35.1,36.1, 40.3, 42.7, 50.1, 53.7, 81.3, 112.1, 114.0, 184.1, 192.4; m/z314.1 (M+1).

Compound 39:

A mixture of compound 38 (Barton et al., 1980) (350 mg, 0.74 mmol) andK₂CO₃ (514 mg, 3.72 mmol) in MeOH (5 mL) was stirred at room temperaturefor 4 h. Ether was added, and the mixture was washed with water. Thecombined aqueous phases were extracted with CH₂Cl₂. The combined organicextracts were dried with MgSO₄ and concentrated. The residue obtainedwas dissolved in CH₂Cl₂ (10 mL). NaOAc (183 mg, 2.23 mmol) and PCC (322mg, 1.49 mmol) were added at room temperature. After stirring for 2 h,additional PCC (160 mg, 0.74 mmol) was added and stirred for another 1h. A mixture of hexanes/EtOAc (1:1, 20 mL) was added and stirred for 5min. The brown slurry was filtered through a pad of silica gel, and thefiltrate was concentrated. The residue obtained was purified by columnchromatography (silica gel, 25% to 33% EtOAc in hexanes) to givecompound 39 (300 mg, 95%) as white solid. ¹H NMR (400 MHz, CDCl₃) δ 5.73(d, 1H, J=1.6 Hz), 4.39 (m, 1H), 3.49 (ddd, 1H, J=2.0, 4.0, 10.8 Hz),3.34 (dd, 1H, J=10.8, 10.8 Hz), 2.17-2.60 (m, 7H), 2.04-2.10 (m, 2H),1.40-1.85 (m, 12H), 1.27 (s, 3H), 1.14 (m, 1H), 1.10 (d, 3H, J=7.2 Hz),0.94 (s, 3H), 0.79 (d, 3H, J=6.8 Hz); m/z 427.2 (M+1).

Compound H0001:

Freshly prepared LDA solution (1.0 M, 0.18 mL, 0.18 mmol) was addeddropwise to a solution of compound 39 (50 mg, 0.12 mmol) in THF (1 mL)at −78° C. After stirring for 45 min, TsCN (43 mg, 0.24 mmol) in THF(0.5 mL) was added. After stirring for another 30 min, NH₄Cl (aq)solution was added to quench the reaction. The reaction mixture wasadjusted to pH 3 using 3 N HCl (aq) and extracted with EtOAc. Thecombined EtOAc extracts were washed with water, dried with MgSO₄, andconcentrated. The crude product obtained was dissolved in benzene (1mL), and DDQ (25 mg, 0.13 mmol) was added. The red solution was refluxedfor 20 min and then cooled to room temperature. The reaction mixture waswashed with NaHCO₃ (aq) solution and water, then dried with MgSO₄ andconcentrated. The residue obtained was purified by column chromatography(silica gel, 33% EtOAc in hexanes) to give H0001 (19 mg, 37%): ¹H NMR(400 MHz, CDCl₃) δ 7.96 (s, 1H), 5.84 (d, 1H, J=2.0 Hz), 4.41 (m, 1H),3.50 (ddd, 1H, J=2.4, 4.0, 11.2 Hz), 3.35 (d, 1H, J=10.8, 11.2 Hz), 2.61(m, 1H), 2.48-2.52 (m, 2H), 2.42 (dd, 1H, J=7.2, 8.8 Hz), 2.13-2.27 (m,3H), 1.51-1.88 (m, 9H), 1.44 (m, 1H), 1.39 (s, 3H), 1.20 (m, 1H), 1.11(d, 3H, J=7.2 Hz), 0.97 (s, 3H), 0.80 (d, 3H, J=6.4 Hz); ¹³C NMR (100MHz, CDCl₃) δ 13.1, 15.0, 17.1, 19.0, 26.3, 28.7, 30.1, 31.3, 31.3,31.7, 36.4, 39.5, 40.9, 42.5, 43.1, 50.9, 52.2, 53.6, 66.9, 79.4, 109.4,113.7, 116.4, 120.6, 162.1, 164.1, 190.6, 203.0; m/z 450.2 (M+1).

Compound 40:

To a mixture of ethylene glycol (6.4 g, 104 mmol) and camphorsulfonicacid (40 mg, 0.17 mmol) in cyclohexane (25 mL) was addeddehydroepiandrosterone (5.0 g, 17.3 mmol). The suspension was heated atreflux with a Dean-Stark trap for 20 h. After cooling, the mixture wasdiluted with sat. sodium bicarbonate (50 mL) and extracted with EtOAc(2×100 mL). The combined organic extracts were washed with brine, driedover MgSO₄, concentrated, and dried under vacuum to give compound 40(5.7 g, 99%) as a white solid: m/z 333.1 (M+1).

Compound 41:

To a solution of compound 40 (2.60 g, 7.82 mmol) in toluene (50 mL) wasadded 3-methyl-2-butanone (25 mL) followed by aluminum tri-isopropoxide(2.4 g, 11.73 mmol). The mixture was heated at reflux for 4 h. Aftercooling, the mixture was diluted with MTBE (100 mL) and washed with sat.potassium dihydrogen phosphate (50 mL). The aqueous layer was extractedwith MTBE (2×50 mL). The combined organic extracts were washed withwater, brine, dried over MgSO₄, concentrated, and dried under vacuum togive crude yellow solid. The crude product was purified by columnchromatography (10% EtOAc/CH₂Cl₂) to give product 41 (1.87 g, 72%) as awhite solid: m/z 331.0 (M+1).

Compound 42:

To a solution of potassium t-butoxide in THF (57.5 mL of 1 M solution,57.5 mmol) was added t-butanol (28 mL). Compound 41 (3.80 g, 11.50 mmol)was added, and the resulting solution was stirred for 30 minutes. Allylbromide (2.78 g, 23.0 mmol) was added, and the mixture was stirred for 2h. Sat. NH₄Cl (aq) (50 mL) was added, and the mixture was diluted withwater (50 mL) and extracted with MTBE (2×100 mL). The combined organicextracts were washed with brine, dried over MgSO₄, concentrated, anddried under vacuum to give crude product. The crude product was purifiedby column chromatography (10-20% EtOAc/hexane) to give product 42 (3.46g, 73%) as a white solid: m/z 411.1 (M+1).

Compound 43:

To a solution of diisopropylamine (0.54 mL, 3.81 mmol) in THF (5 mL) at−78° C. was added n-BuLi (2.21 mL of a 1.6 M solution in hexane, 3.53mmol). The solution was allowed to warm to 0° C. and stirred for 20 min,then re-cooled to −78° C. A solution of compound 42 (0.58 g, 1.41 mmol)in THF (2 mL) was added dropwise, and the solution was stirred 30 min. Asolution of tosyl cyanide (0.28 g, 1.55 mmol) in THF (2 mL) was addeddropwise, and the solution was stirred 30 min. Water (5 mL) was added,and the mixture was allowed to warm to room temperature. The aqueousphase was adjusted to pH 4 with 1 N HCl (aq) and extracted with MTBE(2×50 mL). The combined organic extracts were washed with brine, driedover MgSO₄, concentrated, and dried under vacuum to give crude product.The crude product was purified by column chromatography (25%EtOAc/hexane) to give compound 43 (0.35 g, 57%) as a white foam solid:m/z 436.1 (M+1).

Compound 44:

A solution of compound 43 (0.34 g, 0.78 mmol) in THF (10 mL) was placedunder N₂ atmosphere. 10% Pd/C (25 mg) was added, and the mixture wasevacuated and purged with H₂ (3×). The mixture was stirred under a H₂balloon for 2 h, then filtered through a fine fitted filter. Thefiltrate was concentrated to give compound 44 (0.34 g, 100%) as a whitefoam solid: m/z 440.2 (M+1).

Compound 63313:

To a solution of compound 44 (0.34 g, 0.77 mmol) in benzene (8 mL) wasadded DDQ (176 mg, 0.77 mmol), and the solution was heated at 80° C. for3 h. After cooling, the mixture was diluted with EtOAc (50 mL), washedwith sat. NaHCO₃ (aq), brine, dried over MgSO₄, and concentrated to givecrude product as a dark foam. The crude product was purified by columnchromatography (15-25% EtOAc/hexane) to give impure product 63313 (95mg) as an off-white foam solid, which was taken up in THF (4 mL) and 1MHCl (1 mL) and stirred 2 h. The mixture was diluted with sat. NaHCO₃(aq), and extracted with EtOAc (2×50 mL). The combined organic extractswere washed with brine, dried over MgSO₄, concentrated, and dried undervacuum to give crude product. The crude product was purified by columnchromatography (15% EtOAc/hexane) to give compound 63313 (34 mg, 10%) asa white foam solid: ¹H NMR (500 MHz, CDCl₃) δ 7.67 (s, 1H), 5.59 (br s,1H), 2.53 (dd, J=19 and 9 Hz, 1H), 2.44 (br d, J=19 Hz, 1H), 2.20-2.08(m, 1H), 2.05-1.35 (m, 14H), 1.30 (s, 3H), 1.30-1.08 (m, 3H), 1.05-0.90(m, 1H), 0.96 (s, 3H), 0.89 (t, J=7 Hz, 3H), 0.85 (t, J=7 Hz, 3H); m/z394.1 (M+1).

Compound 43:

To a solution of compound 42 (0.87 g, 2.12 mmol) in CH₂Cl₂ (100 mL) wasadded the 2^(nd) generation Grubbs catalyst (90 mg, 0.11 mmol), and thesolution was stirred at room temperature for 2 h. Most of the CH₂Cl₂ wasremoved via rotary evaporation. The crude product was purified by columnchromatography (15% EtOAc/hexane) to give compound 43 (0.80 g, 99%) as awhite crystalline solid: m/z 383.0 (M+1).

Compound 44:

To a solution of di-isopropylamine (0.39 mL, 2.73 mmol) in THF (5 mL) at−78° C. was added n-BuLi (1.58 mL of a 1.6 M solution in hexane, 2.53mmol). The solution was allowed to warm to 0° C. and stirred for 20 min,then re-cooled to −78° C. A solution of compound 43 (0.387 g, 1.01 mmol)in THF (15 mL) and toluene (10 mL) was added dropwise, and the solutionwas stirred 30 min. A solution of tosyl cyanide (0.22 g, 1.21 mmol) inTHF (3 mL) was added dropwise, and the solution was stirred 30 min.Water (5 mL) was added, and the mixture was allowed to warm to roomtemperature. The aqueous phase was adjusted to pH 4 with 1 N HCl (aq)and extracted with MTBE (2×50 mL). The combined organic extracts werewashed with brine, dried over MgSO₄, concentrated, and dried undervacuum. The crude product was purified by column chromatography (20%EtOAc/hexane) to give compound 44 (0.14 g, 34%) as a white foam solid:m/z 408.1 (M+1).

Compound 63304:

To a solution of compound 44 (0.11 g, 0.27 mmol) in benzene (3 mL) wasadded DDQ (61 mg, 0.27 mmol). The solution was heated at 80° C. for 30min. After cooling, the mixture was diluted with MTBE (50 mL), washedwith sat. NaHCO₃ (20 mL), brine, dried over MgSO₄, and concentrated togive crude product as a light brown foam. The crude product was purifiedby column chromatography (0.5-1% EtOAc/CH₂Cl₂) to give product 63304 (20mg, 18%) as a white foam solid: ¹H NMR (500 MHz, CDCl₃) δ 7.65 (s, 1H),5.78 (br s, 1H), 5.68 (br s, 1H), 5.51 (br s, 1H), 4.00-3.85 (m, 4H),2.88 (d, J=16 Hz, 1H), 2.82 (d, J=16 Hz, 1H), 2.71 (d, J=16 Hz, 1H),2.61 (d, J=16 Hz, 1H), 2.22-2.15 (m, 1H), 2.05-1.97 (m, 1H), 1.90-1.40(m, 9H), 1.30 (s, 3H), 1.33-1.22 (m, 2H), 0.86 (s, 3H); m/z 406.1 (M+1).

Compound 63311:

To a solution of compound 63304 (0.030 g, 0.074 mmol) in THF (5 mL) andwater (1 mL) was added 1 N HCl (1 mL). The solution was stirredovernight and then allowed to stand for 72 h. The mixture was dilutedwith sat. NaHCO₃ (aq), and extracted with EtOAc (2×25 mL). The combinedorganic extracts were washed with brine, dried over MgSO₄, concentrated,and dried under vacuum to give crude product. The crude product waspurified by column chromatography (15% EtOAc/hexane) to give product63311 (17 mg, 64%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 7.65 (s,1H), 5.80 (m, 1H), 5.73 (m, 1H), 5.53 (m, 1H), 2.91 (d, J=16 Hz, 1H),2.84 (d, J=16 Hz, 1H), 2.74 (br d, J=16 Hz, 1H), 2.62 (br d, J=16 Hz,1H), 2.50 (dd, J=19 and 9 Hz, 1H), 2.32 (dt, J=18 and 5 Hz, 1H), 2.13(dt, J=19 and 9 Hz, 1H), 2.03-1.75 (m, 5H), 1.73-1.53 (m, 2H), 1.43-1.28(m, 3H), 1.33 (s, 3H), 0.94 (s, 3H); m/z 362.0 (M+1).

Compound 45:

A solution of compound 43 (0.79 g, 2.07 mmol) in THF (25 mL) was placedunder N₂ atmosphere. 5% Pd/C (100 mg) was added, and the mixture wasevacuated and purged with H₂ (3×). The mixture was stirred under a H₂balloon for 2 h, then filtered through a fine frit. The filtrate wasconcentrated and dried under vacuum to give compound 45 (0.78 g, 98%) asa white solid: m/z 385.1 (M+1).

Compound 46:

To a solution of di-isopropylamine (0.77 mL, 5.48 mmol) in THF (10 mL)at −78° C. was added n-BuLi (3.17 mL of a 1.6 M solution in hexane, 5.07mmol). The solution was allowed to warm to 0° C. and stirred for 20 min,then re-cooled to −78° C. A solution of compound 45 (0.78 g, 2.03 mmol)in THF (10 mL) was added dropwise, and the solution was stirred 30 min.A solution of tosyl cyanide (0.44 g, 2.43 mmol) in THF (5 mL) was addeddropwise, and the solution was stirred 30 min. Sat. NH₄Cl (aq) (10 mL)was added, and the mixture was allowed to warm to room temperature. Themixture was diluted with water (10 mL) and extracted with MTBE (2×50mL). The combined organic extracts were washed with brine, dried overMgSO₄, concentrated, and dried under vacuum to give to give crudeproduct. The crude product was purified by column chromatography (15-20%EtOAc/hexane) to give compound 46 (0.46 g, 55%) as a white foam solid:m/z 410.1 (M+H).

Compound 63317:

A solution of compound 46 (0.34 g, 0.83 mmol) in DMF (3 mL) was cooledin an ice bath. 1,3-Dibromo-5,5-dimethylhydantoin (0.142 g, 0.50 mmol)was added, and the solution was stirred 2 h at 0° C. Pyridine (0.75 mL)was added, and the solution was heated at 55° C. for 21 h. Aftercooling, the mixture was diluted with water (10 mL) and extracted withMTBE (2×30 mL). The combined organic extracts were washed with 0.5 M HCl(aq) (2×15 mL), sat. NaHCO₃ (aq) (20 mL), brine, dried over MgSO₄,concentrated, and dried under vacuum to give crude product. The crudeproduct was purified by column chromatography (20% EtOAc/hexane) to giveproduct 63317 (0.258 g, 76%) as a white foam solid: ¹H NMR (500 MHz,CDCl₃) δ 7.63 (s, 1H), 5.69 (br s, 1H), 3.98-3.83 (m, 4H), 2.28-2.12 (m,2H), 2.10-1.96 (m, 2H), 1.94-1.40 (m, 15H), 1.35 (s, 3H), 1.34-1.20 (m,2H), 0.91 (s, 3H); m/z 408.1 (M+1).

Compound 63318:

To a solution of 63317 (0.23 g, 0.56 mmol) in THF (10 mL) was added 0.5N HCl (aq) (3 mL). The solution was stirred 50 h. The mixture wasdiluted with sat. NaHCO₃ (aq) and extracted with MTBE (2×50 mL). Thecombined organic extracts were washed with brine, dried over MgSO₄,concentrated, and dried under vacuum at 50° C. to give product 63318(200 mg, 98%) as a white foam solid: ¹H NMR (500 MHz, CDCl₃) δ 7.62 (s,1H), 5.73 (br s, 1H), 2.50 (dd, J=19 and 9 Hz, 1H), 2.34 (dt, J=19 and 5Hz, 1H), 2.22-2.02 (m, 2H), 2.01-1.55 (m, 15H), 1.38 (s, 3H), 1.38-1.24(m, 2H), 0.94 (s, 3H); m/z 364.1 (M+1).

Compound 63329:

To a solution of compound 63318 (0.069 g, 0.19 mmol) in toluene (1 mL)and isopropanol (1 mL) was added aluminum tri-isopropoxide (58 mg, 0.28mmol). The mixture was stirred at 75° C. for 20 h. The mixture wasdiluted with EtOAc (50 mL), washed with 1 N HCl (aq) (20 mL), sat.NaHCO₃ (aq) (20 mL), brine, dried over MgSO₄, and concentrated to givecrude product. This crude product was combined with that from a previousrun (0.082 mmol scale) and was purified by column chromatography (10-20%EtOAc/CH₂Cl₂) to give product 63329 (20 mg, 20%) as a white foam solid:¹H NMR (500 MHz, CDCl₃) δ 7.61 (s, 1H), 5.71 (br s, 1H), 3.70 (br t, J=8Hz, 1H), 2.29-2.06 (m, 3H), 1.98-1.13 (m, 18H), 1.38 (s, 3H), 1.08-0.97(m, 1H), 0.83 (s, 3H); m/z 366.0 (M+1).

Compound 47:

A solution of acetyl chloride (0.125 mL) in 125 mL of methanol wastreated with cholic acid (5 g, 0.012 mol) in one portion. The solutionwas stirred at room temperature for 72 h, at which time the solvent wasconcentrated in vacuo to give compound 47 (5.0 g, quantitative) as whitesolid: m/z 423.2 (M+1).

Compound 48:

Compound 47 (5.0 g, 0.0118 mol) was suspended in toluene (150 mL),followed by the addition of silver carbonate on celite (6.5 g) in oneportion. The reaction mixture was refluxed for 3 hrs and then filteredhot through a sintered glass funnel. The filtrate was concentrated invacuo giving a crude product. The crude product was taken up inCH₂Cl₂/THF (7:3) and passed through a pad of silica gel, eluting withCH₂Cl₂/THF (7:3), to give compound 48 (3.3 g, 66%) as a white solid: m/z421.1 (M+1).

Compound 49:

Compound 48 (3.3 g, 7.85 mmol) was diluted with 25 mL of CH₂Cl₂,followed by the addition of diisopropylethylamine (3.3 mL) andchloromethyl methylether (1.89 g, 23.5 mmol), respectively. The solutionwas stirred at 45° C. for overnight (˜14 h), cooled to room temperature,and was then concentrated to give a viscous liquid. This crude productwas taken up in EtOAc (50 mL) and washed with a sat. KH₂PO₄ (aq)solution and brine. The organic phase was dried over Na₂SO₄, filtered,and the filtrate was concentrated in vacuo to give crude product. Thecrude product was purified by column chromatography (elution withhexane/EtOAc 7:3) to give compound 49 (2.8 g, 70%) as a viscouscolorless liquid which solidified on standing: m/z 385.1 (M+1).

Compound 50:

Compound 49 (0.62 g, 1.21 mmol) was suspended in 9 mL of ethyl formate,followed by the dropwise addition of 30% sodium methoxide in methanol(0.5 mL). The reaction mixture was stirred at room temperature forapproximately 2 h, at which time degassing was complete. To the reactionmixture was added 0.1 N HCl (aq) until the solution was at pH=3. Ethylacetate (40 mL) and water (40 mL) were added, the organic phase wasseparated and dried over MgSO₄. The drying agent was filtered, and thefiltrate was concentrated to give crude product. The crude product waspurified by column chromatography (elution with hexane/EtOAc 7:3) togive product 50 (0.37 g) as a white solid: m/z 551.2 (M+1).

Compound 51:

Compound 50 (0.37 g, 0.67 mmol) was suspended in an ethanol/watersolution (30 mL/5 mL), followed by the addition of hydroxylaminehydrochloride (0.12 g) in one portion. The solution was stirred at 60°C. for overnight (˜14 h), cooled to room temperature, and concentratedin vacuo. The product was partitioned between CH₂Cl₂ (20 mL) and brine(20 mL). The organic phase was separated, dried over MgSO₄, thenfiltered, and the filtrate was concentrated to give crude product. Thecrude product was purified by column chromatography (elution withhexane/EtOAc 7:3) to give compound 51 (0.15 g, 40% from 49) as a viscousliquid: m/z 548.3 (M+1).

Compound 52:

Compound 51 (0.15 g, 0.27 mmol) was diluted with 1:1 THF/methanol (5mL), followed by the dropwise addition of 30% sodium methoxide inmethanol (0.5 mL). The reaction mixture was stirred at 55° C. for 2hours, cooled to room temperature, and was then diluted with 1:1EtOAc:1N HCl (aq) (40 mL). The organic phase was separated, dried(Na₂SO₄), filtered, and the filtrate was concentrated to dryness to givecrude product. The crude product was purified by column chromatography(elution with hexane/EtOAc 7:3) to give compound 52 (0.060 g, 41%) as awhite solid: m/z 440.1 (M+1).

Compound 63312:

Compound 52 (0.050 g, 0.093 mmol) was taken up in DMF (1 mL), followedby the addition of 1,3-dibromo-5,5-dimethylhydantoin (0.016 g, 0.055mmol). The solution was stirred at room temperature for 3 hours, thentreated with 0.25 mL of anhydrous pyridine. The solution was heated to55° C. and was stirred at that temperature for 3 days. The solution wascooled to room temperature and diluted with EtOAc and 1 N HCl (aq) (25mL each). The organic phase was separated, washed with water, and driedover sodium sulfate. The drying agent was filtered, and the filtrate wasconcentrated in vacuo to give crude product. The crude product waspurified by column chromatography (gradient elution with hexane/EtOAc,from 7:3 to 1:1) to give product 63312 (0.040 g, 80%) as a whiteamorphous solid: ¹H NMR (500 MHz, CDCl₃) δ 7.55 (s, 1H), 4.70 (d, J=7Hz, 1H), 4.67 (d, J=7 Hz, 1H), 4.64 (d, J=7 Hz, 1H), 4.56 (d, J=7 Hz,1H), 3.77 (br s, 1H), 3.72 (br s, 1H), 3.66 (s, 3H), 3.48 (t, J=16 Hz,1H), 3.39 (s, 3H), 3.30 (s, 3H), 2.45-2.31 (m, 2H), 2.23 (m, 1H),2.15-2.02 (m, 2H), 1.92-1.53 (m, 11H), 1.44-1.23 (m, 2H), 1.25 (s, 3H),1.08 (m, 1H), 0.92 (d, J=6 Hz, 3H), 0.73 (s, 3H); m/z 438, 408, and 376.

Compound 63314:

Compound 63312 (0.025 g, 0.047 mmol) was taken up in 2 mL of CH₂Cl₂ andthen treated with 1 mL of 2 M HCl in diethyl ether. The solution wasstirred at room temperature for overnight, then concentrated to dryness.The residue was was purified by column chromatography (gradient elutionwith 7:3 to 1:1 hexane:EtOAc) to give product 63314 (0.010 g, 48%) as aviscous colorless liquid: ¹H NMR (500 MHz, CDCl₃) δ 7.52 (s, 1H), 4.06(br s, 1H), 3.95 (br s, 1H), 3.59 (s, 3H), 3.54 (dd, J=17 and 15 Hz,1H), 2.47 (dd, J=18 and 4 Hz, 1H), 2.26 (m, 1H), 2.10 (m, 1H), 2.03-1.53(m, 14H), 1.50-1.13 (m, 4H), 1.27 (s, 3H), 1.00 (d, J=6 Hz, 3H), 0.76(s, 3H); m/z 444.1 (M+1).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A compound comprising: a) the basic skeleton of an organic compoundhaving two to eight five and/or six-membered rings provided that thebasic skeleton is not the basic skeleton of argentatin, betulinic acid,lanostane, oleanic acid, boswellic acid, glycyrrhetinic acid, ursolicacid, or tricyclic-bis-enone; b) a structural unit of the formula:

 wherein: the carbon atoms labeled 1, 2 and 3 are part of a five orsix-membered ring; X is cyano or —C(O)R_(a), wherein R_(a) is: hydrogen,hydroxy, halo, amino, hydroxyamino, azido or mercapto; oralkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)), aryl_((C≦12)),aralkyl_((C≦12)), heteroaryl_((C≦12)), heteroaralkyl_((C≦12)),alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),heteroaralkoxy_((C≦12)), acyloxy_((C≦12)), alkylamino_((C≦12)),dialkylamino_((C≦12)), alkoxyamino_((C≦12)), alkenylamino_((C≦12)),alkynylamino_((C≦12)), arylamino_((C≦12)), aralkylamino_((C≦12)),heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),alkylsulfonylamino_((C≦12)), amido_((C≦12)), alkyl-silyloxy_((C≦12)), orsubstituted versions of any of these groups; and R₁ and R₂ are eachindependently: hydrogen; or alkyl_((C≦12)), alkenyl_((C≦12)),alkynyl_((C≦12)), aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),heteroaralkoxy_((C≦12)), acyloxy_((C≦12)), alkylamino_((C≦12)),dialkylamino_((C≦12)), arylamino_((C≦12)), aralkylamino_((C≦12)),heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)), amido_((C≦12)),or a substituted version of any of these groups; or R₁ and R₂ are takentogether and are alkanediyl_((C≦12)), alkenediyl_((C≦12)),alkanediyl_((C≦12)) or alkenediyl_((C≦12)); and c) from 0 to 8 chemicalgroups attached to a carbon atom of the basic skeleton other than carbonatoms 1, 2, 3 or 4, wherein each chemical group is independently:hydroxy, halo, oxo, amino, hydroxyamino, nitro, imino, cyano, azido,mercapto, or thio; or alkyl_((C≦12)), alkenyl_((C≦12)),alkynyl_((C≦12)), aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),heteroaralkyl_((C≦12)), acyl_((C≦12)), alkylidene_((C≦12)),alkoxy_((C≦12)), alkenyloxy_((C≦12)), alkynyloxy_((C≦12)),aryloxy_((C≦12)), aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),heteroaralkoxy_((C≦12)), acyloxy_((C≦12)), alkylamino_((C≦12)),dialkylamino_((C≦12)), alkoxyamino_((C≦12)), alkenylamino_((C≦12)),alkynylamino_((C≦12)), arylamino_((C≦12)), aralkylamino_((C≦12)),heteroarylamino_((C≦12)), heteroaralkylamino_((C≦12)),alkylsulfonylamino_((C≦12)), amido_((C≦12)), alkylimino_((C≦12)),alkenylimino_((C≦12)), alkynylimino_((C≦12)), arylimino_((C≦12)),aralkylimino_((C≦12)), heteroarylimino_((C≦12)),heteroaralkylimino_((C≦12)), acylimino_((C≦12)), alkylthio_((C≦12)),alkenylthio_((C≦12)), alkynylthio_((C≦12)), arylthio_((C≦12)),aralkylthio_((C≦12)), heteroarylthio_((C≦12)),heteroaralkylthio_((C≦12)), acylthio_((C≦12)), thioacyl_((C≦12)),alkylsulfonyl_((C≦12)), alkenylsulfonyl_((C≦12)),alkynylsulfonyl_((C≦12)), arylsulfonyl_((C≦12)),aralkylsulfonyl_((C≦12)), heteroarylsulfonyl_((C≦12)),heteroaralkylsulfonyl_((C≦12)), alkyl-ammonium_((C≦12)),alkylsulfonium_((C≦12)), alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), ora substituted version of any of these groups; or pharmaceuticallyacceptable salts, esters, hydrates, solvates, tautomers, acetals,ketals, prodrugs, or optical isomers thereof. 2-173. (canceled)